Compositions comprising HMW-MAA and fragments thereof, and methods of use thereof

ABSTRACT

This invention provides recombinant polypeptides comprising a fragment of a High Molecular Weight Melanoma-Associated Antigen (HMW-MAA), recombinant  Listeria  strains comprising same, and methods of inducing an immune response and treating and impeding the growth of tumors, comprising administering same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser. No. 60/837,608 filed Aug. 15, 2006, which is incorporated in its entirety herein by reference.

FIELD OF INVENTION

This invention provides recombinant polypeptides comprising a fragment of a High Molecular Weight Melanoma-Associated Antigen (HMW-MAA), recombinant Listeria strains comprising same, and methods of inducing an immune response and treating and impeding the growth of tumors, comprising administering same.

BACKGROUND OF THE INVENTION

HMW-MAA, also known as the melanoma chondroitin sulfate proteoglycan (MCSP), is a transmembrane protein of 2322 residues. HMW-MAA is expressed on over 90% of surgically removed benign nevi and melanoma lesions, and is also expressed in basal cell carcinoma, tumors of neural crest origin (e.g. astrocytomas, gliomas, neuroblastomas and sarcomas), childhood leukemias, and lobular breast carcinoma lesions.

Treatments and cures for many tumors e.g. HMW-MAA-expressing tumors, as well as methods for prevention especially in high risk populations, are urgently needed in the art.

SUMMARY OF THE INVENTION

This invention provides in some embodiments, recombinant polypeptides comprising a fragment of a High Molecular Weight-Melanoma Associated Antigen (HMW-MAA), recombinant Listeria strains comprising same, and methods of inducing an immune response and treating and impeding the growth of tumors, comprising administering same.

In one embodiment, the present invention provides a recombinant Listeria strain comprising a recombinant polypeptide, the recombinant polypeptide comprising a fragment of a HMW-MAA protein (“HMW-MAA fragment”).

In another embodiment, the present invention provides a recombinant polypeptide comprising a fragment of a HMW-MAA protein operatively linked to a non-HMW-MAA oligopeptide selected from a listeriolysin (LLO) oligopeptide, an ActA oligopeptide, or a PEST-like oligopeptide or homologues thereof.

In another embodiment, the present invention provides a recombinant polypeptide comprising a fragment of a HMW-MAA protein, wherein the fragment consists of about amino acids (AA) 360-554 of the HMW-MAA protein from which the fragment is derived. In another embodiment, the fragment consists of about AA 701-1130. In another embodiment, the fragment consists of about AA 2160-2258. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a vaccine comprising a recombinant Listeria strain of the present invention and an adjuvant.

In another embodiment, the present invention provides an immunogenic composition comprising a recombinant Listeria strain of the present invention.

In another embodiment, the present invention provides a vaccine comprising a recombinant polypeptide of the present invention.

In another embodiment, the present invention provides an immunogenic composition comprising a recombinant polypeptide of the present invention.

In another embodiment, the present invention provides a recombinant vaccine vector encoding a recombinant polypeptide of the present invention.

In another embodiment, the present invention provides a recombinant Listeria strain comprising a recombinant polypeptide of the present invention.

In another embodiment, the present invention provides a method of inducing an anti-HMW-MAA immune response in a subject, comprising administering to the subject a composition comprising a recombinant Listeria strain of the present invention, thereby inducing an anti-HMW-MAA immune response in a subject.

In another embodiment, the present invention provides a method of delaying progression of a solid tumor in a subject, comprising administering to the subject a composition comprising a recombinant Listeria strain of the present invention, thereby delaying progression of a solid tumor in a subject. In another embodiment, the subject mounts an immune response against a pericyte of the solid tumor. In another embodiment, the pericyte is in a vasculature of the solid tumor. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of impeding vascularization of a solid tumor in a subject, comprising administering to the subject a composition comprising a recombinant Listeria strain of the present invention, thereby impeding vascularization of a solid tumor in a subject. In another embodiment, the subject mounts an immune response against a pericyte of the solid tumor. In another embodiment, the pericyte is in a vasculature of the solid tumor. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of delaying progression of a HMW-MAA-expressing tumor in a subject, comprising administering to the subject a composition comprising a recombinant Listeria strain of the present invention, thereby delaying progression of a HMW-MAA-expressing tumor in a subject. In another embodiment, the subject mounts an immune response against the HMW-MAA-expressing tumor. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of treating a HMW-MAA-expressing tumor in a subject, comprising administering to the subject a composition comprising a recombinant Listeria strain of the present invention, thereby treating a HMW-MAA-expressing tumor in a subject. In another embodiment, the subject mounts an immune response against the HMW-MAA-expressing tumor. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of inducing an anti-HMW-MAA immune response in a subject, comprising administering to the subject a composition comprising a recombinant polypeptide of the present invention, thereby inducing an anti-HMW-MAA immune response in a subject.

In another embodiment, the present invention provides a method of delaying progression of a solid tumor in a subject, comprising administering to the subject a composition comprising a recombinant polypeptide of the present invention, thereby delaying progression of a solid tumor in a subject. In another embodiment, the subject mounts an immune response against a pericyte of the solid tumor. In another embodiment, the pericyte is in a vasculature of the solid tumor. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of impeding a vascularization of a solid tumor in a subject, comprising administering to the subject a composition comprising a recombinant polypeptide of the present invention, thereby impeding a vascularization of a solid tumor in a subject. In another embodiment, the subject mounts an immune response against a pericyte of the solid tumor. In another embodiment, the pericyte is in a vasculature of the solid tumor. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of delaying progression of a HMW-MAA-expressing tumor in a subject, comprising administering to the subject a composition comprising a recombinant polypeptide of the present invention, thereby delaying progression of a HMW-MAA-expressing tumor in a subject. In another embodiment, the subject mounts an immune response against the HMW-MAA-expressing tumor. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of treating a HMW-MAA-expressing tumor in a subject, comprising administering to the subject a composition comprising a recombinant polypeptide of the present invention, thereby treating a HMW-MAA-expressing tumor in a subject. In another embodiment, the subject mounts an immune response against the HMW-MAA-expressing tumor. Each possibility represents a separate embodiment of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: HMW-MAA cloning into pGG55-based plasmid. Lm-LLO-HMW-MAA was generated by transforming the prfA⁻ strain XFL-7 with the plasmid pGG-55. pGG-55 has the hly promoter driving expression of a non-hemolytic fusion of LLO-E7 and the prfA gene to select for retention of the plasmid. XFL-7 must retain the plasmid in order to be viable.

FIG. 2. LLO-HMW-MAA constructs are expressed. Supernatant was harvested from LM strains transformed with the LLO-HMW-MAA A, B and C plasmids. A. Anti-PEST probes revealed that all three strains produced fusion proteins of the expected sizes (48 Kda for LLO, 75 Kda for HMW-MAA-A, 98 Kda for HMW-MAA-B, and 62 Kda for HMW-MAA-C). B. Anti-LLO probes revealed LLO bands for HMW-MAA-A, HMW-MAA-B, HMW-MAA-C, and in 10403S controls.

FIG. 3. Listeria strains expressing LLO-HMW-MAA constructs exhibit growth in media (A), virulence, and intracellular growth (B) similar to wild type Lm.

FIG. 4. HMW-MAA-expressing Lm impedes the growth of tumors, even in tumor cells that do not express HMW-MAA. 10⁸ cfu of Lm-HMW-MAA-C impedes B 16F0-Ova tumor growth as measured by tumor size (A) and volume (B) significantly compared to the naïve group. Similar effects on tumor diameter and volume were observed with all three Lm-LLO-HMW-MAA strains after inoculation of C57BL/6 mice with B16F0-Ova (C) and RENCA (D and E) tumor cells.

FIG. 5. Selection of HMW-MAA-expressing B16F10 murine tumor cell clones.

FIG. 6. Lm-HMW-MAA constructs induced antigen-specific immune responses that impede tumor growth.

FIG. 7. In vivo depletion of either CD4+ or CD8+ cells abrogated the efficacy of Lm-HMW-MAA-C vaccine.

FIG. 8. CD8+ T cells from mice vaccinated with Lm-HMW-MAA-C mice inhibited the growth of B16F10 HMW-MAA tumors in vivo.

FIG. 9. Mice vaccinated with Lm-HMW-MAA-C that eliminated the B 16F10-HMW-MAA tumor were protected against a second challenge with the same tumor.

FIG. 10. Immunization of HLA-A2/Kb transgenic mice with Lm-HMW-MAA-B and Lm-HMW-MAA-C induced detectable immune responses against two characterized HMW-MAA HLA-A2 epitopes in fragments B and C.

FIG. 11. IFN-γ secretion by T cells stimulated with an HLA-A2 restricted peptide from fragment C of HMW-MAA after one immunization with Lm-HMW-MAA-C in HLA-A2/Kb and wild-type C57B1/6 mice.

FIG. 12. Depiction of vaccinia virus constructs expressing different forms of HPV16E7 protein.

FIG. 13. Induction and penetration of E7 specific CD8⁺ T cells in the spleens and tumors of mice administered TC-1 cells and subsequently administered a recombinant Listeria vaccine (naive, Lm-LLO-E7, Lm-E7, Lm-ActA-E7).

FIG. 14. A. Listeria constructs containing PEST regions lead to greater tumor regression. B. average tumor size in mice treated with Listeria vaccines.

FIG. 15. Listeria constructs containing PEST regions induce a higher percentage of E7-specific lymphocytes in the spleen. A. data from 1 representative experiment. B. average and SE of data from 3 experiments.

FIG. 16. Listeria constructs containing PEST regions induce a higher percentage of E7-specific lymphocytes within the tumor. A. data from 1 representative experiment. B. average and SE of data from 3 experiments.

FIG. 17. Depiction of vaccinia virus constructs expressing different forms of HPV16E7 protein.

FIG. 18. VacLLOE7 induces long-term regression of tumors established from 2×10⁵ TC-1 cells in C57BL/6 mice. Mice were injected 11 and 18 days after tumor challenge with 10⁷ PFU of VacLLOE7, VacSigE7LAMP-1, or VacE7/mouse i.p. or were left untreated (naive). 8 mice per treatment group were used, and the cross section for each tumor (average of 2 measurements) is shown for the indicated days after tumor inoculation.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides recombinant polypeptides comprising a fragment of a High Molecular Weight Melanoma-Associated Antigen (HMW-MAA), recombinant Listeria strains comprising same, and methods of inducing an immune response and treating and impeding the growth of tumors, comprising administering same.

In one embodiment, the present invention provides a recombinant Listeria strain comprising a recombinant polypeptide, the recombinant polypeptide comprising a fragment of a HMW-MAA protein (“HMW-MAA fragment”). In another embodiment, a recombinant Listeria strain of the present invention expresses a recombinant polypeptide of the present invention. In another embodiment, a recombinant Listeria strain of the present invention comprises an isolated nucleic acid that encodes a recombinant polypeptide of the present invention. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a recombinant polypeptide comprising a fragment of a HMW-MAA protein operatively linked to a non-HMW-MAA oligopeptide selected from a listeriolysin (LLO) oligopeptide, an ActA oligopeptide, or a PEST-like oligopeptide or a fragment thereof. In one embodiment, the fragment has the same or a similar properties or function as the full length peptide or protein, as may be demonstrated using assays and tools known in the art. Properties and functions of full length peptides and proteins of the present invention are described in detail hereinbelow.

In another embodiment, the present invention provides a recombinant polypeptide comprising a fragment of a HMW-MAA protein, wherein the fragment consists of about amino acids (AA) 360-554 of the HMW-MAA protein from which the fragment is derived. In another embodiment, the fragment consists of about AA 701-1130. In another embodiment, the fragment has a sequence selected from SEQ ID No: 21-23. In another embodiment, the fragment consists of about AA 2160-2258. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a recombinant polypeptide comprising a fragment of a HMW-MAA protein with an amino acid sequence as set forth in SEQ ID No: 21.

In another embodiment, the present invention provides a recombinant polypeptide comprising a fragment of a HMW-MAA protein with an amino acid sequence as set forth in SEQ ID No: 22.

In another embodiment, the present invention provides a recombinant polypeptide comprising a fragment of a HMW-MAA protein with an amino acid sequence as set forth in SEQ ID No: 23.

In another embodiment, a recombinant polypeptide of the present invention further comprises a non-HMW-MAA peptide. In another embodiment, the non-HMW-MAA peptide enhances the immunogenicity of the fragment. Each possibility represents a separate embodiment of the present invention.

The non-HMW-MAA peptide is, in another embodiment, a listeriolysin (LLO) oligopeptide. In another embodiment, the non-HMW-MAA peptide is an ActA oligopeptide. In another embodiment, the non-HMW-MAA peptide is a PEST-like oligopeptide. As provided herein, fusion to LLO, ActA, PEST-like sequences and fragments thereof enhances the cell-mediated immunogenicity of antigens. In one embodiment, fusion to LLO, ActA, PEST-like sequences and fragments thereof enhances the cell-mediated immunogenicity of antigens in a variety of expression systems. In one embodiment, the expression system is viral, while in another embodiment, the expression system is bacterial. In another embodiment, the non-HMW-MAA peptide is any other immunogenic non-HMW-MAA peptide known in the art. Each possibility represents a separate embodiment of the present invention.

An LLO oligopeptide of methods and compositions of the present invention is, in another embodiment, a non-hemolytic LLO oligopeptide. In another embodiment, the oligopeptide is an LLO fragment. In another embodiment, the oligopeptide is a complete LLO protein. In another embodiment, the oligopeptide is any LLO protein or fragment thereof known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the LLO fragment is rendered non-hemolytic by chemical treatment. In another embodiment, the chemical treatment comprises glutaraldehyde. In another embodiment, the chemical treatment comprises a similarly acting compound. In another embodiment, the chemical treatment comprises any other suitable compound known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the LLO protein utilized to construct vaccines of the present invention has the following sequence:

MKKIMLVFITILVSLPIAQQTEAKDASAFNKENSISSMAPPASPPASPKTPIEKKHADEIDKYIQ GLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQVVNAISSLTYPG ALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVERWN EKYAQAYPNVSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQEEVISFKQ IYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKVK AAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETP GVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNYDPEGN EIVQHKNWSENNKSKLAHFTSSIYLPGNARNINVYAKECTGLAWEWWRTVIDDRNLPLVKNR NISIWGTTLYPKYSNKVDNPIE (GenBank Accession No. P13128; SEQ ID NO: 1; the nucleic acid sequence is set forth in GenBank Accession No. X15127). In one embodiment, the first 25 AA of the proprotein corresponding to this sequence are the signal sequence and are cleaved from LLO when it is secreted by the bacterium. Thus, according to this embodiment, the full length active LLO protein is 504 residues long. In another embodiment, the above sequence is used as the source of the LLO fragment incorporated in a vaccine of the present invention. In another embodiment, an LLO AA sequence of methods and compositions of the present invention is a homologue of SEQ ID No: 1. In another embodiment, the LLO AA sequence is a variant of SEQ ID No: 1. In another embodiment, the LLO AA sequence is a fragment of SEQ ID No: 1. In another embodiment, the LLO AA sequence is an isoform of SEQ ID No: 1. Each possibility represents a separate embodiment of the present invention.

In one embodiment, an isoform is a peptide or protein that has the same function and a similar or identical sequence to another peptide or protein, but is the product of a different gene. In one embodiment, a variant is a peptide or protein that differs from another a peptide or protein in a minor way.

In another embodiment, an LLO protein fragment is utilized in compositions and methods of the present invention. In another embodiment, the LLO fragment is an N-terminal fragment. In another embodiment, the N-terminal LLO fragment has the sequence:

MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSVAPPASPPASPKTPIEKKHADEIDKYIQ GLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQVVNAISSLTYP GALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVER WNEKYAQAYSNVSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQEEVIS FKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGRQVYLKLSTNSHST KVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDLKKGATFN RETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNYD (SEQ ID NO: 2). In another embodiment, an LLO AA sequence of methods and compositions of the present invention comprises the sequence set forth in SEQ ID No: 2. In another embodiment, the LLO AA sequence is a homologue of SEQ ID No: 2. In another embodiment, the LLO AA sequence is a variant of SEQ ID No: 2. In another embodiment, the LLO AA sequence is a fragment of SEQ ID No: 2. In another embodiment, the LLO AA sequence is an isoform of SEQ ID No: 2. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the LLO fragment has the sequence:

MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSVAPPASPPASPKTPIEKKHADEIDKYIQ GLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQVVNAISSLTYP GALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVER WNEKYAQAYSNVSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQEEVI SFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGRQVYLKLSTNSH STKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGA TFNRETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTD (SEQ ID NO: 3). In another embodiment, an LLO AA sequence of methods and compositions of the present invention comprises the sequence set forth in SEQ ID No: 3. In another embodiment, the LLO AA sequence is a homologue of SEQ ID No: 3. In another embodiment, the LLO AA sequence is a variant of SEQ ID No: 3. In another embodiment, the LLO AA sequence is a fragment of SEQ ID No: 3. In another embodiment, the LLO AA sequence is an isoform of SEQ ID No: 3. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the LLO fragment of methods and compositions of the present invention comprises a PEST-like domain. In another embodiment, an LLO fragment that comprises a PEST sequence is utilized as part of a composition or in the methods of the present invention.

In another embodiment, the LLO fragment does not contain the activation domain at the carboxy terminus. In another embodiment, the LLO fragment does not include cysteine 484. In another embodiment, the LLO fragment is a non-hemolytic fragment. In another embodiment, the LLO fragment is rendered non-hemolytic by deletion or mutation of the activation domain. In another embodiment, the LLO fragment is rendered non-hemolytic by deletion or mutation of cysteine 484. In another embodiment, an LLO sequence is rendered non-hemolytic by deletion or mutation at another location.

In another embodiment, the LLO fragment consists of about the first 441 AA of the LLO protein. In another embodiment, the LLO fragment comprises about the first 400-441 AA of the 529 AA full length LLO protein. In another embodiment, the LLO fragment corresponds to AA 1-441 of an LLO protein disclosed herein. In another embodiment, the LLO fragment consists of about the first 420 AA of LLO. In another embodiment, the LLO fragment corresponds to AA 1-420 of an LLO protein disclosed herein. In another embodiment, the LLO fragment consists of about AA 20-442 of LLO. In another embodiment, the LLO fragment corresponds to AA 20-442 of an LLO protein disclosed herein. In another embodiment, any ΔLLO without the activation domain comprising cysteine 484, and in particular without cysteine 484, are suitable for methods and compositions of the present invention.

In another embodiment, the LLO fragment corresponds to the first 400 AA of an LLO protein. In another embodiment, the LLO fragment corresponds to the first 300 AA of an LLO protein. In another embodiment, the LLO fragment corresponds to the first 200 AA of an LLO protein. In another embodiment, the LLO fragment corresponds to the first 100 AA of an LLO protein. In another embodiment, the LLO fragment corresponds to the first 50 AA of an LLO protein, which in one embodiment, comprises one or more PEST-like sequences.

In another embodiment, the LLO fragment contains residues of a homologous LLO protein that correspond to one of the above AA ranges. The residue numbers need not, in another embodiment, correspond exactly with the residue numbers enumerated above; e.g. if the homologous LLO protein has an insertion or deletion, relative to an LLO protein utilized herein.

Each LLO protein and LLO fragment represents a separate embodiment of the present invention.

In another embodiment, homologues of LLO from other species, including known lysins, or fragments thereof may be used as the non-HMW-MAA.

In another embodiment of methods and compositions of the present invention, a fragment of an ActA protein is fused to the HMW-MAA fragment. In another embodiment, the fragment of an ActA protein has the sequence:

MRAMMVVFITANCITINPDIIFAATDSEDSSLNTDEWEEEKTEEQPSEVNTGPRYETAR EVSSRDIKELEKSNKVRNTNKADLIAMLKEKAEKGPNINNNNSEQTENAAINEEASGADRPAI QVERRHPGLPSDSAAEIKKRRKAIASSDSELESLTYPDKPTKVNKKKVAKESVADASESDLDS SMQSADESSPQPLKANQQPFFPKVFKKIKDAGKWVRDKIDENPEVKKAIVDKSAGLIDQLLTK KKSEEVNASDFPPPPTDEELRLALPETPMLLGFNAPATSEPSSFEFPPPPTDEELRLALPETPMLL GFNAPATSEPSSFEFPPPPTEDELEIIRETASSLDSSFTRGDLASLRNAINRHSQNFSDFPPIPTEEE LNGRGGRP (SEQ ID No: 4). In another embodiment, an ActA AA sequence of methods and compositions of the present invention comprises the sequence set forth in SEQ ID No: 4. In another embodiment, the ActA AA sequence is a homologue of SEQ ID No: 4. In another embodiment, the ActA AA sequence is a variant of SEQ ID No: 4. In another embodiment, the ActA AA sequence is a fragment of SEQ ID No: 4. In another embodiment, the ActA AA sequence is an isoform of SEQ ID No: 4. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the ActA fragment is encoded by a recombinant nucleotide comprising the sequence:

ATGCGTGCGATGATGGTGGTTTTCATTACTGCCAATTGCATTACGATTAACCCCGACATAA TATTTGCAGCGACAGATAGCGAAGATTCTAGTCTAAACACAGATGAATGGGAAGAAGAAA AAACAGAAGAGCAACCAAGCGAGGTAAATACGGGACCAAGATACGAAACTGCACGTGAA GTAAGTTCACGTGATATTAAAGAACTAGAAAAATCGAATAAAGTGAGAAATACGAACAAA GCAGACCTAATAGCAATGTTGAAAGAAAAAGCAGAAAAAGGTCCAAATATCAATAATAAC AACAGTGAACAAACTGAGAATGCGGCTATAAATGAAGAGGCTTCAGGAGCCGACCGACCA GCTATACAAGTGGAGCGTCGTCATCCAGGATTGCCATCGGATAGCGCAGCGGAAATTAAAA AAAGAAGGAAAGCCATAGCATCATCGGATAGTGAGCTTGAAAGCCTTACTTATCCGGATAA ACCAACAAAAGTAAATAAGAAAAAAGTGGCGAAAGAGTCAGTTGCGGATGCTTCTGAAAG TGACTTAGATTCTAGCATGCAGTCAGCAGATGAGTCTTCACCACAACCTTTAAAAGCAAAC CAACAACCATTTTTCCCTAAAGTATTTAAAAAAATAAAAGATGCGGGGAAATGGGTACGTG ATAAAATCGACGAAAATCCTGAAGTAAAGAAAGCGATTGTTGATAAAAGTGCAGGGTTAA TTGACCAATTATTAACCAAAAAGAAAAGTGAAGAGGTAAATGCTTCGGACTTCCCGCCACC ACCTACGGATGAAGAGTTAAGACTTGCTTTGCCAGAGACACCAATGCTTCTTGGTTTTAAT GCTCCTGCTACATCAGAACCGAGCTCATTCGAATTTCCACCACCACCTACGGATGAAGAGT TAAGACTTGCTTTGCCAGAGACGCCAATGCTTCTTGGTTTTAATGCTCCTGCTACATCGGAA CCGAGCTCGTTCGAATTTCCACCGCCTCCAACAGAAGATGAACTAGAAATCATCCGGGAA ACAGCATCCTCGCTAGATTCTAGTTTTACAAGAGGGGATTTAGCTAGTTTGAGAAATGCTA TTAATCGCCATAGTCAAAATTTCTCTGATTTCCCACCAATCCCAACAGAAGAAGAGTTGAA CGGGAGAGGCGGTAGACCA (SEQ ID NO: 5). In another embodiment, the recombinant nucleotide has the sequence set forth in SEQ ID NO: 5. In another embodiment, an ActA-encoding nucleotide of methods and compositions of the present invention comprises the sequence set forth in SEQ ID No: 5. In another embodiment, the ActA-encoding nucleotide is a homologue of SEQ ID No: 5. In another embodiment, the ActA-encoding nucleotide is a variant of SEQ ID No: 5. In another embodiment, the ActA-encoding nucleotide is a fragment of SEQ ID No: 5. In another embodiment, the ActA-encoding nucleotide is an isoform of SEQ ID No: 5. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the ActA fragment is any other ActA fragment known in the art. In another embodiment, a recombinant nucleotide of the present invention comprises any other sequence that encodes a fragment of an ActA protein. In another embodiment, the recombinant nucleotide comprises any other sequence that encodes an entire ActA protein. Each possibility represents a separate embodiment of the present invention.

In another embodiment of methods and compositions of the present invention, a PEST-like AA sequence is fused to the HMW-MAA fragment. In another embodiment, the PEST-like AA sequence is KENSISSMAPPASPPASPKTPIEKKHADEIDK (SEQ ID NO: 6). In another embodiment, the PEST-like sequence is KENSISSMAPPASPPASPK (SEQ ID No: 7). In another embodiment, fusion of an antigen to any LLO sequence that includes the 1 of the PEST-like AA sequences enumerated herein can enhance cell mediated immunity against HMW-MAA.

In another embodiment, the PEST-like AA sequence is a PEST-like sequence from a Listeria ActA protein. In another embodiment, the PEST-like sequence is KTEEQPSEVNTGPR (SEQ ID NO: 8), KASVTDTSEGDLDSSMQSADESTPQPLK (SEQ ID NO: 9), KNEEVNASDFPPPPTDEELR (SEQ ID NO: 10), or RGGIPTSEEFSSLNSGDFTDDENSETTEEEIDR (SEQ ID NO: 11). In another embodiment, the PEST-like sequence is from Listeria seeligeri cytolysin, encoded by the lso gene. In another embodiment, the PEST-like sequence is RSEVTISPAETPESPPATP (SEQ ID NO: 12). In another embodiment, the PEST-like sequence is from Streptolysin O protein of Streptococcus sp. In another embodiment, the PEST-like sequence is from Streptococcus pyogenes Streptolysin O, e.g. KQNTASTETTTTNEQPK (SEQ ID NO: 13) at AA 35-51. In another embodiment, the PEST-like sequence is from Streptococcus equisimilis Streptolysin O, e.g. KQNTANTETTTTNEQPK (SEQ ID NO: 14) at AA 38-54. In another embodiment, the PEST-like sequence has a sequence selected from SEQ ID NO: 8-14. In another embodiment, the PEST-like sequence has a sequence selected from SEQ ID NO: 6-14. In another embodiment, the PEST-like sequence is another PEST-like AA sequence derived from a prokaryotic organism.

Identification of PEST-like sequences is well known in the art, and is described, for example in Rogers S et al (Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. Science 1986; 234(4774):364-8) and Rechsteiner M et al (PEST sequences and regulation by proteolysis. Trends Biochem Sci 1996; 21(7):267-71). “PEST-like sequence” refers, in another embodiment, to a region rich in proline (P), glutamic acid (E), serine (S), and threonine (T) residues. In another embodiment, the PEST-like sequence is flanked by one or more clusters containing several positively charged amino acids. In another embodiment, the PEST-like sequence mediates rapid intracellular degradation of proteins containing it. In another embodiment, the PEST-like sequence fits an algorithm disclosed in Rogers et al. In another embodiment, the PEST-like sequence fits an algorithm disclosed in Rechsteiner et al. In another embodiment, the PEST-like sequence contains one or more internal phosphorylation sites, and phosphorylation at these sites precedes protein degradation.

In one embodiment, PEST-like sequences of prokaryotic organisms are identified in accordance with methods such as described by, for example Rechsteiner and Rogers (1996, Trends Biochem. Sci. 21:267-271) for LM and in Rogers S et al (Science 1986; 234(4774):364-8). Alternatively, PEST-like AA sequences from other prokaryotic organisms can also be identified based on this method. Other prokaryotic organisms wherein PEST-like AA sequences would be expected to include, but are not limited to, other Listeria species. In one embodiment, the PEST-like sequence fits an algorithm disclosed in Rogers et al. In another embodiment, the PEST-like sequence fits an algorithm disclosed in Rechsteiner et al. In another embodiment, the PEST-like sequence is identified using the PEST-find program.

In another embodiment, identification of PEST motifs is achieved by an initial scan for positively charged AA R, H, and K within the specified protein sequence. All AA between the positively charged flanks are counted and only those motifs are considered further, which contain a number of AA equal to or higher than the window-size parameter. In another embodiment, a PEST-like sequence must contain at least 1 P, 1 D or E, and at least 1 S or T.

In another embodiment, the quality of a PEST motif is refined by means of a scoring parameter based on the local enrichment of critical AA as well as the motif's hydrophobicity. Enrichment of D, E, P, S and T is expressed in mass percent (w/w) and corrected for 1 equivalent of D or E, 1 of P and 1 of S or T. In another embodiment, calculation of hydrophobicity follows in principle the method of J. Kyte and R. F. Doolittle (Kyte, J and Dootlittle, R F. J. Mol. Biol. 157,105 (1982). For simplified calculations, Kyte-Doolittle hydropathy indices, which originally ranged from −4.5 for arginine to +4.5 for isoleucine, are converted to positive integers, using the following linear transformation, which yielded values from 0 for arginine to 90 for isoleucine. Hydropathy index=10*Kyte-Doolittle hydropathy index+45

In another embodiment, a potential PEST motif's hydrophobicity is calculated as the sum over the products of mole percent and hydrophobicity index for each AA species. The desired PEST score is obtained as combination of local enrichment term and hydrophobicity term as expressed by the following equation: PEST score=0.55*DEPST−0.5*hydrophobicity index.

In another embodiment, “PEST-like sequence” or “PEST-like sequence peptide” refers to a peptide having a score of at least +5, using the above algorithm. In another embodiment, the term refers to a peptide having a score of at least 6. In another embodiment, the peptide has a score of at least 7. In another embodiment, the score is at least 8. In another embodiment, the score is at least 9. In another embodiment, the score is at least 10. In another embodiment, the score is at least 11. In another embodiment, the score is at least 12. In another embodiment, the score is at least 13. In another embodiment, the score is at least 14. In another embodiment, the score is at least 15. In another embodiment, the score is at least 16. In another embodiment, the score is at least 17. In another embodiment, the score is at least 18. In another embodiment, the score is at least 19. In another embodiment, the score is at least 20. In another embodiment, the score is at least 21. In another embodiment, the score is at least 22. In another embodiment, the score is at least 22. In another embodiment, the score is at least 24. In another embodiment, the score is at least 24. In another embodiment, the score is at least 25. In another embodiment, the score is at least 26. In another embodiment, the score is at least 27. In another embodiment, the score is at least 28. In another embodiment, the score is at least 29. In another embodiment, the score is at least 30. In another embodiment, the score is at least 32. In another embodiment, the score is at least 35. In another embodiment, the score is at least 38. In another embodiment, the score is at least 40. In another embodiment, the score is at least 45. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the PEST-like sequence is identified using any other method or algorithm known in the art, e.g the CaSPredictor (Garay-Malpartida H M, Occhiucci J M, Alves J, Belizario J E. Bioinformatics. 2005 June; 21 Suppl 1:i169-76). In another embodiment, the following method is used:

A PEST index is calculated for each stretch of appropriate length (e.g. a 30-35 AA stretch) by assigning a value of 1 to the AA Ser, Thr, Pro, Glu, Asp, Asn, or Gln. The coefficient value (CV) for each of the PEST residue is 1 and for each of the other AA (non-PEST) is 0.

Each method for identifying a PEST-like sequence represents a separate embodiment of the present invention.

In another embodiment, the PEST-like sequence is any other PEST-like sequence known in the art. Each PEST-like sequence and type thereof represents a separate embodiment of the present invention.

“Fusion to a PEST-like sequence” refers, in another embodiment, to fusion to a protein fragment comprising a PEST-like sequence. In another embodiment, the term includes cases wherein the protein fragment comprises surrounding sequence other than the PEST-like sequence. In another embodiment, the protein fragment consists of the PEST-like sequence. Thus, in another embodiment, “fusion” refers to two peptides or protein fragments either linked together at their respective ends or embedded one within the other. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the HMW-MAA fragment of methods and compositions of the present invention is fused to the non-HMW-MAA AA sequence. In another embodiment, the HMW-MAA fragment is embedded within the non-HMW-MAA AA sequence. In another embodiment, an HMW-MAA-derived peptide is incorporated into an LLO fragment, ActA protein or fragment, or PEST-like sequence. Each possibility represents a separate embodiment of the present invention.

In another embodiment, fusion proteins of the present invention are prepared by a process comprising subcloning of appropriate sequences, followed by expression of the resulting nucleotide. In another embodiment, subsequences are cloned and the appropriate subsequences cleaved using appropriate restriction enzymes. The fragments are then ligated, in another embodiment, to produce the desired DNA sequence. In another embodiment, DNA encoding the fusion protein is produced using DNA amplification methods, for example polymerase chain reaction (PCR). First, the segments of the native DNA on either side of the new terminus are amplified separately. The 5′ end of the one amplified sequence encodes the peptide linker, while the 3′ end of the other amplified sequence also encodes the peptide linker. Since the 5′ end of the first fragment is complementary to the 3′ end of the second fragment, the two fragments (after partial purification, e.g. on LMP agarose) can be used as an overlapping template in a third PCR reaction. The amplified sequence will contain codons, the segment on the carboxy side of the opening site (now forming the amino sequence), the linker, and the sequence on the amino side of the opening site (now forming the carboxyl sequence). The insert is then ligated into a plasmid. In another embodiment, a similar strategy is used to produce a protein wherein an HMW-MAA fragment is embedded within a heterologous peptide.

In one embodiment, LLO sequences fused to a HMW-MAA fragment such as A, B, or C or Listeria expressing a HMW-MAA fragment increased the immune response to said peptide (Example 5), conferred antitumor immunity (Examples 4 and 5), and generated peptide-specific IFN-gamma-secreting CD8+ cells (Example 5). In one embodiment, ActA, LLO and/or PEST-like sequences fused to a peptide such as HPV E7 increased the immune response to said peptide, conferred antitumor immunity, and generated peptide-specific IFN-gamma-secreting CD8+ cells (Examples 6 and 7), even when the fusion peptide was expressed in a non-Listeria vector (Example 8).

In another embodiment, a recombinant polypeptide of the present invention is made by a process comprising the step of chemically conjugating a first polypeptide comprising an HMW-MAA fragment to a second polypeptide comprising a non-HMW-MAA peptide. In another embodiment, an HMW-MAA fragment is conjugated to a second polypeptide comprising the non-HMW-MAA peptide. In another embodiment, a peptide comprising an HMW-MAA fragment is conjugated to a non-HMW-MAA peptide. In another embodiment, an HMW-MAA fragment is conjugated to a non-HMW-MAA peptide. Each possibility represents a separate embodiment of the present invention.

The HMW-MAA protein from which HMW-MAA fragments of the present invention are derived is, in another embodiment, a human HMW-MAA protein. In another embodiment, the HMW-MAA protein is a mouse protein. In another embodiment, the HMW-MAA protein is a rat protein. In another embodiment, the HMW-MAA protein is a primate protein. In another embodiment, the HMW-MAA protein is from any other species known in the art. In another embodiment, the HMW-MAA protein is melanoma chondroitin sulfate proteoglycan (MCSP). In another embodiment, an AN2 protein is used in methods and compositions of the present invention. In another embodiment, an NG2 protein is used in methods and compositions of the present invention.

In another embodiment, the HMW-MAA protein of methods and compositions of the present invention has the sequence:

MQSGRGPPLPAPGLALALTLTMLARLASAASFFGENHLEVPVATALTDIDLQLQFSTSQ PEALLLLAAGPADHLLLQLYSGRLQVRLVLGQEELRLQTPAETLLSDSIPHTVVLTVVEGWATL SVDGFLNASSAVPGAPLEVPYGLFVGGTGTLGLPYLRGTSRPLRGCLHAATLNGRSLLRPLTPD VHEGCAEEFSASDDVALGFSGPHSLAAFPAWGTQDEGTLEFTLTTQSRQAPLAFQAGGRRGDF IYVDIFEGHLRAVVEKGQGTVLLHNSVPVADGQPHEVSVHINAHRLEISVDQYPTHTSNRGVLS YLEPRGSLLLGGLDAEASRHLQEHRLGLTPEATNASLLGCMEDLSVNGQRRGLREALLTRNMA AGCRLEEEEYEDDAYGHYEAFSTLAPEAWPAMELPEPCVPEPGLPPVFANFTQLLTISPLVVAE GGTAWLEWRHVQPTLDLMEAELRKSQVLFSVTRGARHGELELDIPGAQARKMFTLLDVVNR KARFIHDGSEDTSDQLVLEVSVTARVPMPSCLRRGQTYLLPIQVNPVNDPPHIIFPHGSLMVILE HTQKPLGPEVFQAYDPDSACEGLTFQVLGTSSGLPVERRDQPGEPATEFSCRELEAGSLVYVH RGGPAQDLTFRVSDGLQASPPATLKVVAIRPAIQIHRSTGLRLAQGSAMPILPANLSVETNAVG QDVSVLFRVTGALQFGELQKQGAGGVEGAEWWATQAFHQRDVEQGRVRYLSTDPQHHAYD TVENLALEVQVGQEILSNLSFPVTIQRATVWMLRLEPLHTQNTQQETLTTAHLEATLEEAGPSPP TFHYEVVQAPRKGNLQLQGTRLSDGQGFTQDDIQAGRVTYGATARASEAVEDTFRFRVTAPPY FSPLYTFPIHIGGDPDAPVLTNVLLVVPEGGEGVLSADHLFVKSLNSASYLYEVMERPRHGRLA WRGTQDKTTMVTSFTNEDLLRGRLVYQHDDSETTEDDIPFVATRQGESSGDMAWEEVRGVFR VAIQPVNDHAPVQTISRIFHVARGGRRLLTTDDVAFSDADSGFADAQLVLTRKDLLFGSIVAVD EPTRPIYRFTQEDLRKRRVLFVHSGADRGWIQLQVSDGQHQATALLEVQASEPYLRVANGSSL VVPQGGQGTIDTAVLHLDTNLDIRSGDEVHYHVTAGPRWGQLVRAGQPATAFSQQDLLDGAV LYSHNGSLSPRDTMAFSVEAGPVHTDATLQVTIALEGPLAPLKLVRHKKIYVFQGEAAEIRRDQ LEAAQEAVPPADIVFSVKSPPSAGYLVMVSRGALADEPPSLDPVQSFSQEAVDTGRVLYLHSRP EAWSDAFSLDVASGLGAPLEGVLVELEVLPAAIPLEAQNFSVPEGGSLTLAPPLLRVSGPYFPTL LGLSLQVLEPPQHGALQKEDGPQARTLSAFSWRMVEEQLIRYVHDGSETLTDSFVLMANASEM DRQSHPVAFTVTVLPVNDQPPILTTNTGLQMWEGATAPIPAEALRSTDGDSGSEDLVYTIEQPS NGRVVLRGAPGTEVRSFTQAQLDGGLVLFSHRGTLDGGFRFRLSDGEHTSPGHFFRVTAQKQV LLSLKGSQTLTVCPGSVQPLSSSQTLRASSSAGTDPQLLLYRVVRGPQLGRLFHAQQDSTGEALV NFTQAEVYAGNILYEHEMPPEPFWEAHDTLELQLSSPPARDVAATLAVAVSFEAACPQRPSHL WKNKGLWVPEGQRARITVAALDASNLLASVPSPQRSEHDVLFQVTQFPSRGQLLVSEEPLHAG QPHFLQSQLAAGQLVYAHGGGGTQQDGFHFRAHLQGPAGASVAGPQTSEAFAITVRDVNERP PQPQASVPLRLTRGSRAPISRAQLSVVDPDSAPGEIEYEVQRAPHNGFLSLVGGGLGPVTRFTQA DVDSGRLAFVANGSSVAGIFQLSMSDGASPPLPMSLAVDILPSAIEVQLRAPLEVPQALGRSSLS QQQLRVVS DREEPEAAYRLIQGPQYGHLLVGGRPTSAFSQFQIDQGEVVFAFTNFSSSHDHFRV LALARGVNASAVVNVTVRALLHVWAGGPWPQGATLRLDPTVLDAGELANRTGSVPRFRLLE GPRHGRVVRVPRARTEPGGSQLVEQFTQQDLEDGRLGLEVGRPEGRAPGPAGDSLTLELWAQ GVPPAVASLDFATEPYNAARPYSVALLSVPEAARTEAGKPESSTPTGEPGPMASSPEPAVAKG GFLSFLEANMFSVIIPMCLVLLLLALILPLLFYLRKRNKTGKHDVQVLTAKPRNGLAGDTETFR KVEPGQAIPLTAVPGQGPPPGGQPDPELLQFCRTPNPALKNGQYWV (SEQ ID No: 15). In another embodiment, an HMW-MAA AA sequence of methods and compositions of the present invention comprises the sequence set forth in SEQ ID No: 15. In another embodiment, the HMW-MAA AA sequence is a homologue of SEQ ID No: 15. In another embodiment, the HMW-MAA AA sequence is a variant of SEQ ID No: 15. In another embodiment, the HMW-MAA AA sequence is a fragment of SEQ ID No: 15. In another embodiment, the HMW-MAA AA sequence is an isoform of SEQ ID No: 15. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the HMW-MAA protein of methods and compositions of the present invention is encoded by the sequence:

atgcagtccggccgcggccccccacttccagcccccggcctggccttggctttgaccctgactatgttggccagacttgcatccgcggcttccttcttcg gtgagaaccacctggaggtgcctgtggccacggctctgaccgacatagacctgcagctgcagttctccacgtcccagcccgaagccctccttctcctg gcagcaggcccagctgaccacctcctgctgcagctctactctggacgcctgcaggtcagacttgttctgggccaggaggagctgaggctgcagactc cagcagagacgctgctgagtgactccatcccccacactgtggtgctgactgtcgtagagggctgggccacgttgtcagtcgatgggtttctgaacgcct cctcagcagtcccaggagcccccctagaggtcccctatgggctctttgttgggggcactgggacccttggcctgccctacctgaggggaaccagccg acccctgaggggttgcctccatgcagccaccctcaatggccgcagcctcctccggcctctgacccccgatgtgcatgagggctgtgctgaagagttttc tgccagtgatgatgtggccctgggcttctctgggccccactctctggctgccttccctgcctggggcactcaggacgaaggaaccctagagtttacactc accacacagagccggcaggcacccttggccttccaggcagggggccggcgtggggacttcatctatgtggacatatttgagggccacctgcgggcc gtggtggagaagggccagggtaccgtattgctccacaacagtgtgcctgtggccgatgggcagccccatgaggtcagtgtccacatcaatgctcaccg gctggaaatctccgtggaccagtaccctacgcatacttcgaaccgaggagtcctcagctacctggagccacggggcagtctccttctcggggggctgg atgcagaggcctctcgtcacctccaggaacaccgcctgggcctgacaccagaggccaccaatgcctccctgctgggctgcatggaagacctcagtgtc aatggccagaggcgggggctgcgggaagctttgctgacgcgcaacatggcagccggctgcaggctggaggaggaggagtatgaggacgatgccta tggacattatgaagctttctccaccctggcccctgaggcttggccagccatggagctgcctgagccatgcgtgcctgagccagggctgcctcctgtcttt gccaatttcacccagctgctgactatcagcccactggtggtggccgaggggggcacagcctggcttgagtggaggcatgtgcagcccacgctggacct gatggaggctgagctgcgcaaatcccaggtgctgttcagcgtgacccgaggggcacgccatggcgagctcgagctggacatcccgggagcccagg cacgaaaaatgttcaccctcctggacgtggtgaaccgcaaggcccgcttcatccacgatggctctgaggacacctccgaccagctggtgctggaggtg tcggtgacggctcgggtgcccatgccctcatgccttcggaggggccaaacatacctcctgcccatccaggtcaaccctgtcaatgacccaccccacatc atcttcccacatggcagcctcatggtgatcctggaacacacgcagaagccgctggggcctgaggttttccaggcctatgacccggactctgcctgtgag ggcctcaccttccaggtccttggcacctcctctggcctccccgtggagcgccgagaccagcctggggagccggcgaccgagttctcctgccgggagtt ggaggccggcagcctagtctatgtccaccgcggtggtcctgcacaggacttgacgttccgggtcagcgatggactgcaggccagccccccggccac gctgaaggtggtggccatccggccggccatacagatccaccgcagcacagggttgcgactggcccaaggctctgccatgcccatcttgcccgccaac ctgtcggtggagaccaatgccgtggggcaggatgtgagcgtgctgttccgcgtcactggggccctgcagtttggggagctgcagaagcagggggcag gtggggtggagggtgctgagtggtgggccacacaggcgttccaccagcgggatgtggagcagggccgcgtgaggtacctgagcactgacccacagc accacgcttacgacaccgtggagaacctggccctggaggtgcaggtgggccaggagatcctgagcaatctgtccttcccagtgaccatccagagagc cactgtgtggatgctgcggctggagccactgcacactcagaacacccagcaggagaccctcaccacagcccacctggaggccaccctggaggaggc aggcccaagccccccaaccttccattatgaggtggttcaggctcccaggaaaggcaaccttcaactacagggcacaaggctgtcagatggccagggc ttcacccaggatgacatacaggctggccgggtgacctatggggccacagcacgtgcctcagaggcagtcgaggacaccttccgtttccgtgtcacagc tccaccatatttctccccactctataccttccccatccacattggtggtgacccagatgcgcctgtcctcaccaatgtcctcctcgtggtgcctgagggtgg tgagggtgtcctctctgctgaccacctctttgtcaagagtctcaacagtgccagctacctctatgaggtcatggagcggcccgccatgggaggttggct tggcgtgggacacaggacaagaccactatggtgacatccttcaccaatgaagacctgttgcgtggccggctggtctaccagcatgatgactccgagac cacagaagatgatatcccatttgttgctacccgccagggcgagagcagtggtgacatggcctgggaggaggtacggggtgtcttccgagtggccatcc agcccgtgaatgaccacgccctgtgcagaccatcagccggatcttccatgtggccggggtgggcggcggctgctgactacagacgacgtggcctt cagcgatgctgactcgggctttgctgacgcccagctggtgcttacccgcaaggacctcctctttggcagtatcgtggccgtagatgagcccacgcggcc catctaccgcttcacccaggaggacctcaggaagaggcgagtactgttcgtgcactcaggggctgaccgtggctggatccagctgcaggtgtccgacg ggcaacaccaggccactgcgctgctggaggtgcaggcctcggaaccctacctccgtgtggccaacggctccagccttgtggtccctcaagggggcc agggcaccatcgacacggccgtgctccacctggacaccaacctcgacatccgcagtggggatgaggtccactaccacgtcacagctggccctcgctg gggacagctagtccgggctggtcagccagccacagccttctcccagcaggacctgctggatggggccgttctctatagccacaatggcagcctcagcc cccgcgacaccatggccttctccgtggaagcagggccagtgcacacggatgccaccctacaagtgaccattgccctagagggcccactggccccact gaagctggtccggcacaagaagatctacgtcttccagggagaggcagctgagatcagaagggaccagctggaggcagcccaggaggcagtgccac ctgcagacatcgtattctcagtgaagagcccaccgagtgccggctacctggtgatggtgtcgcgtggcgccttggcagatgagccacccagcctggac cctgtgcagagcttctcccaggaggcagtggacacaggcagggtcctgtacctgcactcccgccctgaggcctggagcgatgccttctcgctggatgt ggcctcaggcctgggtgctcccctcgagggcgtccttgtggagctggaggtgctgcccgctgccatcccactagaggcgcaaaacttcagcgtccctg agggtggcagcctcaccctggcccctccactgctccgtgtctccgggccctacttccccactctcctgggcctcagcctgcaggtgctggagccacccc agcatggagccctgcagaaggaggacggacctcaagccaggaccctcagcgccttctcctggagaatggtggaagagcagctgatccgctacgtgc atgacgggagcgagacactgacagacagttttgtcctgatggctaatgcctccgagatggatcgccagagccatcctgtggccttcactgtcactgtcct gcctgtcaatgaccaaccccccatcctcactacaaacacaggcctgcagatgtgggagggggccactgcgcccatccctgcggaggctctgaggagc acggacggcgactctgggtctgaggatctggtctacaccatcgagcagcccagcaacgggcgggtagtgctgcggggggcgccgggcactgaggt gcgcagcttcacgcaggcccagctggacggcgggctcgtgctgttctcacacagaggaaccctggatggaggcttccgcttccgcctctctgacggc gagcacacttcccccggacacttcttccgagtgacggcccagaagcaagtgctcctctcgctgaagggcagccagacactgactgtctgcccagggtc cgtccagccactcagcagtcagaccctcagggccagctccagcgcaggcactgacccccagctcctgctctaccgtgtggtgcggggcccccagcta ggccggctgttccacgcccagcaggacagcacaggggaggccctggtgaacttcactcaggcagaggtctacgctgggaatattctgtatgagcatg agatgcccccgagcccttttgggaggcccatgataccctagagctccagctgtcctcgccgcctgcccgggacgtggccgccacccttgctgtggct gtgtcttttgaggctgcctgtccccagcgccccagccacctctggaagaacaaaggtctctgggtccccgagggccagcgggccaggatcaccgtgg ctgctctggatgcctccaatctcttggccagcgttccatcaccccagcgctcagagcatgatgtgctcttccaggtcacacagttccccagccggggcca gctgttggtgtccgaggagcccctccatgctgggcagccccacttcctgcagtcccagctggctgcagggcagctagtgtatgcccacggcggtgggg gcacccagcaggatggcttccactttcgtgcccacctccaggggccagcaggggcctccgtggctggaccccaaacctcagaggcctttgccatcac ggtgagggatgtaaatgagcggccccctcagccacaggcctctgtcccactccggctcacccgaggctctcgtgcccccatctcccgggcccagctg agtgtggtggacccagactcagctcctggggagattgagtacgaggtccagcgggcaccccacaacggcttcctcagcctggtgggtggtggcctgg ggcccgtgacccgcttcacgcaagccgatgtggattcagggcggctggccttcgtggccaacgggagcagcgtggcaggcatcttccagctgagcat gtctgatggggccagcccacccctgcccatgtccctggctgtggacatcctaccatccgccatcgaggtgcagctgcgggcacccctggaggtgcccc aagctttggggcgctcctcactgagccagcagcagctccgggtggtttcagatcgggaggagccagaggcagcataccgcctcatccagggacccca gtatgggcatctcctggtgggcgggcggcccacctcggccttcagccaattccagatagaccagggcgaggtggtctttgccttcaccaacttctcctcc tctcatgaccacttcagagtcctggcactggctaggggtgtcaatgcatcagccgtagtgaacgtcactgtgagggctctgctgcatgtgtgggcaggtg ggccatggccccagggtgccaccctgcgcctggaccccaccgtcctagatgctggcgagctggccaaccgcacaggcagtgtgccgcgcttccgcc tcctggagggaccccggcatggccgcgtggtccgcgtgccccgagccaggacggagcccgggggcagccagctggtggagcagttcactcagca ggaccttgaggacgggaggctggggctggaggtgggcaggccagaggggagggcccccggccccgcaggtgacagtctcactctggagctgtgg gcacagggcgtcccgcctgctgtggcctccctggactttgccactgagccttacaatgctgcccggccctacagcgtggccctgctcagtgtccccgag gccgcccggacggaagcagggaagccagagagcagcacccccacaggcgagccaggccccatggcatccagccctgagcccgctgtggccaag ggaggcttcctgagcttccttgaggccaacatgttcagcgtcatcatccccatgtgcctggtacttctgctcctggcgctcatcctgcccctgctcttctacct ccgaaaacgcaacaagacgggcaagcatgacgtccaggtcctgactgccaagccccgcaacggcctggctggtgacaccgagacctttcgcaaggt ggagccaggccaggccatcccgctcacagctgtgcctggccaggggccccctccaggaggccagcctgacccagagctgctgcagttctgccgga cacccaaccctgcccttaagaatggccagtactgggtgtgaggcctggcctgggcccagatgctgatcgggccagggacaggc (SEQ ID No: 16). In another embodiment, the recombinant nucleotide has the sequence set forth in SEQ ID NO: 16. In another embodiment, an HMW-MAA-encoding nucleotide of methods and compositions of the present invention comprises the sequence set forth in SEQ ID No: 16. In another embodiment, the HMW-MAA-encoding nucleotide is a homologue of SEQ ID No: 16. In another embodiment, the HMW-MAA-encoding nucleotide is a variant of SEQ ID No: 16. In another embodiment, the HMW-MAA-encoding nucleotide is a fragment of SEQ ID No: 16. In another embodiment, the HMW-MAA-encoding nucleotide is an isoform of SEQ ID No: 16. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the HMW-MAA protein of methods and compositions of the present invention has an AA sequence set forth in a GenBank entry having an Accession Numbers selected from NM_(—)001897 and X96753. In another embodiment, the HMW-MAA protein is encoded by a nucleotide sequence set forth in one of the above GenBank entries. In another embodiment, the HMW-MAA protein comprises a sequence set forth in one of the above GenBank entries. In another embodiment, the HMW-MAA protein is a homologue of a sequence set forth in one of the above GenBank entries. In another embodiment, the HMW-MAA protein is a variant of a sequence set forth in one of the above GenBank entries. In another embodiment, the HMW-MAA protein is a fragment of a sequence set forth in one of the above GenBank entries. In another embodiment, the HMW-MAA protein is an isoform of a sequence set forth in one of the above GenBank entries. Each possibility represents a separate embodiment of the present invention.

The HMW-MAA fragment utilized in the present invention comprises, in another embodiment, AA 360-554. In another embodiment, the fragment consists essentially of AA 360-554. In another embodiment, the fragment consists of AA 360-554. In another embodiment, the fragment comprises AA 701-1130. In another embodiment, the fragment consists essentially of AA 701-1130. In another embodiment, the fragment consists of AA 701-1130. In another embodiment, the fragment comprises AA 2160-2258. In another embodiment, the fragment consists essentially of 2160-2258. In another embodiment, the fragment consists of 2160-2258. Each possibility represents a separate embodiment of the present invention.

In some embodiments, a polypeptide of the present invention will comprise a fragment of a HMW-MAA protein, in any form or embodiment as described herein. In some embodiments, any of the polypeptides of the present invention will consist of a fragment of a HMW-MAA protein, in any form or embodiment as described herein. In some embodiments, of the compositions of this invention will consist essentially of a fragment of a HMW-MAA protein, in any form or embodiment as described herein. In some embodiments, the term “comprise” refers to the inclusion of the indicated active agent, such as the fragment of a HMW-MAA protein, or the fragment of a HMW-MAA protein and a non-HMW-MAA polypeptide, as well as inclusion of other active agents, and pharmaceutically acceptable carriers, excipients, emollients, stabilizers, etc., as are known in the pharmaceutical industry. In some embodiments, the term “consisting essentially of” refers to a composition, whose only active ingredient is the indicated active ingredient, however, other compounds may be included which are for stabilizing, preserving, etc. the formulation, but are not involved directly in the therapeutic effect of the indicated active ingredient. In some embodiments, the term “consisting essentially of” may refer to components which facilitate the release of the active ingredient. In some embodiments, the term “consisting” refers to a composition, which contains the active ingredient and a pharmaceutically acceptable carrier or excipient.

In another embodiment, the HMW-MAA fragment is approximately 98 AA in length. In another embodiment, the length is approximately 194 AA. In another embodiment, the length is approximately 430 AA. In another embodiment, the length is approximately 98-194 AA. In another embodiment, the length is approximately 194-430 AA. In another embodiment, the length is approximately 98-430 AA.

In another embodiment, the length of the HMW-MAA fragment of the present invention is at least 8 amino acids (AA). In another embodiment, the length is more than 8 AA. In another embodiment, the length is at least 9 AA. In another embodiment, the length is more than 9 AA. In another embodiment, the length is at least 10 AA. In another embodiment, the length is more than 10 AA. In another embodiment, the length is at least 11 AA. In another embodiment, the length is more than 11 AA. In another embodiment, the length is at least 12 AA. In another embodiment, the length is more than 12 AA. In another embodiment, the length is at least about 14 AA. In another embodiment, the length is more than 14 AA. In another embodiment, the length is at least about 16 AA. In another embodiment, the length is more than 16 AA. In another embodiment, the length is at least about 18 AA. In another embodiment, the length is more than 18 AA. In another embodiment, the length is at least about 20 AA. In another embodiment, the length is more than 20 AA. In another embodiment, the length is at least about 25 AA. In another embodiment, the length is more than 25 AA. In another embodiment, the length is at least about 30 AA. In another embodiment, the length is more than 30 AA. In another embodiment, the length is at least about 40 AA. In another embodiment, the length is more than 40 AA. In another embodiment, the length is at least about 50 AA. In another embodiment, the length is more than 50 AA. In another embodiment, the length is at least about 70 AA. In another embodiment, the length is more than 70 AA. In another embodiment, the length is at least about 100 AA. In another embodiment, the length is more than 100 AA. In another embodiment, the length is at least about 150 AA. In another embodiment, the length is more than 150 AA. In another embodiment, the length is at least about 200 AA. In another embodiment, the length is more than 200 AA. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the length is about 8-50 AA. In another embodiment, the length is about 8-70 AA. In another embodiment, the length is about 8-100 AA. In another embodiment, the length is about 8-150 AA. In another embodiment, the length is about 8-200 AA. In another embodiment, the length is about 8-250 AA. In another embodiment, the length is about 8-300 AA. In another embodiment, the length is about 8-400 AA. In another embodiment, the length is about 8-500 AA. In another embodiment, the length is about 9-50 AA. In another embodiment, the length is about 9-70 AA. In another embodiment, the length is about 9-100 AA. In another embodiment, the length is about 9-150 AA. In another embodiment, the length is about 9-200 AA. In another embodiment, the length is about 9-250 AA. In another embodiment, the length is about 9-300 AA. In another embodiment, the length is about 10-50 AA. In another embodiment, the length is about 10-70 AA. In another embodiment, the length is about 10-100 AA. In another embodiment, the length is about 10-150 AA. In another embodiment, the length is about 10-200 AA. In another embodiment, the length is about 10-250 AA. In another embodiment, the length is about 10-300 AA. In another embodiment, the length is about 10-400 AA. In another embodiment, the length is about 10-500 AA. In another embodiment, the length is about 11-50 AA. In another embodiment, the length is about 11-70 AA. In another embodiment, the length is about 11-100 AA. In another embodiment, the length is about 11-150 AA. In another embodiment, the length is about 11-200 AA. In another embodiment, the length is about 11-250 AA. In another embodiment, the length is about 11-300 AA. In another embodiment, the length is about 11-400 AA. In another embodiment, the length is about 11-500 AA. In another embodiment, the length is about 12-50 AA. In another embodiment, the length is about 12-70 AA. In another embodiment, the length is about 12-100 AA. In another embodiment, the length is about 12-150 AA. In another embodiment, the length is about 12-200 AA. In another embodiment, the length is about 12-250 AA. In another embodiment, the length is about 12-300 AA. In another embodiment, the length is about 12-400 AA. In another embodiment, the length is about 12-500 AA. In another embodiment, the length is about 15-50 AA. In another embodiment, the length is about 15-70 AA. In another embodiment, the length is about 15-100 AA. In another embodiment, the length is about 15-150 AA. In another embodiment, the length is about 15-200 AA. In another embodiment, the length is about 15-250 AA. In another embodiment, the length is about 15-300 AA. In another embodiment, the length is about 15-400 AA. In another embodiment, the length is about 15-500 AA. In another embodiment, the length is about 8-400 AA. In another embodiment, the length is about 8-500 AA. In another embodiment, the length is about 20-50 AA. In another embodiment, the length is about 20-70 AA. In another embodiment, the length is about 20-100 AA. In another embodiment, the length is about 20-150 AA. In another embodiment, the length is about 20-200 AA. In another embodiment, the length is about 20-250 AA. In another embodiment, the length is about 20-300 AA. In another embodiment, the length is about 20-400 AA. In another embodiment, the length is about 20-500 AA. In another embodiment, the length is about 30-50 AA. In another embodiment, the length is about 30-70 AA. In another embodiment, the length is about 30-100 AA. In another embodiment, the length is about 30-150 AA. In another embodiment, the length is about 30-200 AA. In another embodiment, the length is about 30-250 AA. In another embodiment, the length is about 30-300 AA. In another embodiment, the length is about 30-400 AA. In another embodiment, the length is about 30-500 AA. In another embodiment, the length is about 40-50 AA. In another embodiment, the length is about 40-70 AA. In another embodiment, the length is about 40-100 AA. In another embodiment, the length is about 40-150 AA. In another embodiment, the length is about 40-200 AA. In another embodiment, the length is about 40-250 AA. In another embodiment, the length is about 40-300 AA. In another embodiment, the length is about 40-400 AA. In another embodiment, the length is about 40-500 AA. In another embodiment, the length is about 50-70 AA. In another embodiment, the length is about 50-100 AA. In another embodiment, the length is about 50-150 AA. In another embodiment, the length is about 50-200 AA. In another embodiment, the length is about 50-250 AA. In another embodiment, the length is about 50-300 AA. In another embodiment, the length is about 50-400 AA. In another embodiment, the length is about 50-500 AA. In another embodiment, the length is about 70-100 AA. In another embodiment, the length is about 70-150 AA. In another embodiment, the length is about 70-200 AA. In another embodiment, the length is about 70-250 AA. In another embodiment, the length is about 70-300 AA. In another embodiment, the length is about 70-400 AA. In another embodiment, the length is about 70-500 AA. In another embodiment, the length is about 100-150 AA. In another embodiment, the length is about 100-200 AA. In another embodiment, the length is about 100-250 AA. In another embodiment, the length is about 100-300 AA. In another embodiment, the length is about 100-400 AA. In another embodiment, the length is about 100-500 AA. Each possibility represents a separate embodiment of the present invention.

Each HMW-MAA protein and each fragment thereof represents a separate embodiment of the present invention.

In another embodiment, a recombinant polypeptide of the methods and compositions of the present invention comprises a signal sequence. In another embodiment, the signal sequence is from the organism used to construct the vaccine vector. In another embodiment, the signal sequence is a LLO signal sequence. In another embodiment, the signal sequence is an ActA signal sequence. In another embodiment, the signal sequence is a Listerial signal sequence. In another embodiment, the signal sequence is any other signal sequence known in the art. Each possibility represents a separate embodiment of the present invention.

The terms “peptide” and “recombinant peptide” refer, in another embodiment, to a peptide or polypeptide of any length. In another embodiment, a peptide or recombinant peptide of the present invention has one of the lengths enumerated above for an HMW-MAA fragment. Each possibility represents a separate embodiment of the present invention. In one embodiment, the term “peptide” refers to native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and/or peptidomimetics (typically, synthetically synthesized peptides), such as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, including, but not limited to, CH2—NH, CH2—S, CH2—S═O, O═C—NH, CH2—O, CH2—CH2, S═C—NH, CH═CH or CF═CH, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinunder.

Peptide bonds (—CO—NH—) within the peptide may be substituted, for example, by N-methylated bonds (—N(CH3)-CO—), ester bonds (—C(R)H—C—O—O—C(R)—N—), ketomethylen bonds (—CO—CH2—), *-aza bonds (—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds (—CH2—NH—), hydroxyethylene bonds (—CH(OH)—CH2—), thioamide bonds (—CS—NH—), olefinic double bonds (—CH═CH—), retro amide bonds (—NH—CO—), peptide derivatives (—N(R)—CH2—CO—), wherein R is the “normal” side chain, naturally presented on the carbon atom.

These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) at the same time. Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted for synthetic non-natural acid such as TIC, naphthylelanine (Nol), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.

In addition to the above, the peptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).

In one embodiment, the term “amino acid” or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term “amino acid” may include both D- and L-amino acids.

Peptides or proteins of this invention may be prepared by various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)).

In one embodiment, the term “oligonucleotide” is interchangeable with the term “nucleic acid”, and may refer to a molecule, which may include, but is not limited to, prokaryotic sequences, eukaryotic mRNA, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. The term also refers to sequences that include any of the known base analogs of DNA and RNA.

In another embodiment, the present invention provides a vaccine comprising a recombinant Listeria strain of the present invention. In one embodiment, the vaccine additionally comprises an adjuvant. In one embodiment, the vaccine additionally comprises a cytokine, chemokine, or combination thereof. In one embodiment, a vaccine is a composition which elicits an immune response to an antigen or polypeptide in the composition as a result of exposure to the composition. In another embodiment, the vaccine or composition additionally comprises APCs, which in one embodiment are autologous, while in another embodiment, they are allogeneic to the subject.

In another embodiment, the present invention provides a vaccine comprising a recombinant polypeptide of the present invention and an adjuvant.

In another embodiment, the present invention provides an immunogenic composition comprising a recombinant polypeptide of the present invention. In another embodiment, the immunogenic composition of methods and compositions of the present invention comprises a recombinant vaccine vector encoding a recombinant peptide of the present invention. In another embodiment, the immunogenic composition comprises a plasmid encoding a recombinant peptide of the present invention. In another embodiment, the immunogenic composition comprises an adjuvant. In one embodiment, a vector of the present invention may be administered as part of a vaccine composition. Each possibility represents a separate embodiment of the present invention.

The immunogenic composition utilized in methods and compositions of the present invention comprises, in another embodiment, a recombinant vaccine vector. In another embodiment, the recombinant vaccine vector comprises a recombinant peptide of the present invention. In another embodiment, the recombinant vaccine vector comprises an isolated nucleic acid of the present invention. In another embodiment, the recombinant vaccine vector comprises an isolated nucleic acid encoding a recombinant peptide of the present invention. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a recombinant vaccine vector encoding a recombinant polypeptide of the present invention. In another embodiment, the present invention provides a recombinant vaccine vector comprising a recombinant polypeptide of the present invention. In another embodiment, the expression vector is a plasmid. Methods for constructing and utilizing recombinant vectors are well known in the art and are described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Brent et al. (2003, Current Protocols in Molecular Biology, John Wiley & Sons, New York). Each possibility represents a separate embodiment of the present invention.

In another embodiment, the vector is an intracellular pathogen. In another embodiment, the vector is derived from a cytosolic pathogen. In another embodiment, the vector is derived from an intracellular pathogen. In another embodiment, an intracellular pathogen induces a predominantly cell-mediated immune response. In another embodiment, the vector is a Salmonella strain. In another embodiment, the vector is a BCG strain. In another embodiment, the vector is a bacterial vector. In another embodiment, dendritic cells transduced with a vector of the present invention may be administered to the subject to upregulate the subject's immune response, which in one embodiment is accomplished by upregulating CTL activity.

In another embodiment, the recombinant vaccine vector induces a predominantly Th1-type immune response.

An immunogenic composition of methods and compositions of the present invention comprises, in another embodiment, an adjuvant that favors a predominantly Th1-type immune response. In another embodiment, the adjuvant favors a predominantly Th1-mediated immune response. In another embodiment, the adjuvant favors a Th1-type immune response. In another embodiment, the adjuvant favors a Th1-mediated immune response. In another embodiment, the adjuvant favors a cell-mediated immune response over an antibody-mediated response. In another embodiment, the adjuvant is any other type of adjuvant known in the art. In another embodiment, the immunogenic composition induces the formation of a T cell immune response against the target protein. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the adjuvant is MPL. In another embodiment, the adjuvant is QS21. In another embodiment, the adjuvant is a TLR agonist. In another embodiment, the adjuvant is a TLR4 agonist. In another embodiment, the adjuvant is a TLR9 agonist. In another embodiment, the adjuvant is Resiquimod®. In another embodiment, the adjuvant is imiquimod. In another embodiment, the adjuvant is a CpG oligonucleotide. In another embodiment, the adjuvant is a cytokine or a nucleic acid encoding same. In another embodiment, the adjuvant is a chemokine or a nucleic acid encoding same. In another embodiment, the adjuvant is IL-12 or a nucleic acid encoding same. In another embodiment, the adjuvant is IL-6 or a nucleic acid encoding same. In another embodiment, the adjuvant is a lipopolysaccharide. In another embodiment, the adjuvant is as described in Fundamental Immunology, 5th ed (August 2003): William E. Paul (Editor); Lippincott Williams & Wilkins Publishers; Chapter 43: Vaccines, G J V Nossal, which is hereby incorporated by reference. In another embodiment, the adjuvant is any other adjuvant known in the art. Each possibility represents a separate embodiment of the present invention. In one embodiment, a “predominantly Th1-type immune response” refers to an immune response in which IFN-gamma is secreted. In another embodiment, it refers to an immune response in which tumor necrosis facto-β is secreted. In another embodiment, it refers to an immune response in which IL-2 is secreted. Each possibility represents a separate embodiment of the present invention. In another embodiment, the vector is selected from Salmonella sp., Shigella sp., BCG, L. monocytogenes (which embodiment is exemplified in Example 2), E. coli, and S. gordonii. In another embodiment, the fusion proteins are delivered by recombinant bacterial vectors modified to escape phagolysosomal fusion and live in the cytoplasm of the cell. In another embodiment, the vector is a viral vector. In other embodiments, the vector is selected from Vaccinia (which embodiment is exemplified in Example 8), Avipox, Adenovirus, AAV, Vaccinia virus NYVAC, Modified vaccinia strain Ankara (MVA), Semliki Forest virus, Venezuelan equine encephalitis virus, herpes viruses, and retroviruses. In another embodiment, the vector is a naked DNA vector. In another embodiment, the vector is any other vector known in the art. Each possibility represents a separate embodiment of the present invention. In another embodiment, the present invention provides an isolated nucleic acid encoding a recombinant polypeptide of the present invention. In one embodiment, the isolated nucleic acid comprises a sequence sharing at least 85% homology with a nucleic acid encoding a recombinant polypeptide of the present invention. In another embodiment, the isolated nucleic acid comprises a sequence sharing at least 90% homology with a nucleic acid encoding a recombinant polypeptide of the present invention. In another embodiment, the isolated nucleic acid comprises a sequence sharing at least 95% homology with a nucleic acid encoding a recombinant polypeptide of the present invention. In another embodiment, the isolated nucleic acid comprises a sequence sharing at least 97% homology with a nucleic acid encoding a recombinant polypeptide of the present invention. In another embodiment, the isolated nucleic acid comprises a sequence sharing at least 99% homology with a nucleic acid encoding a recombinant polypeptide of the present invention.

In another embodiment, the present invention provides a vaccine comprising a recombinant nucleotide molecule of the present invention and an adjuvant. In another embodiment, the present invention provides a recombinant vaccine vector comprising a recombinant nucleotide molecule of the present invention. In another embodiment, the present invention provides a recombinant vaccine vector encoding a recombinant polypeptide of the present invention. In another embodiment, the present invention provides a recombinant vaccine vector comprising a recombinant polypeptide of the present invention. In another embodiment, the expression vector is a plasmid. Methods for constructing and utilizing recombinant vectors are well known in the art and are described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Brent et al. (2003, Current Protocols in Molecular Biology, John Wiley & Sons, New York). Each possibility represents a separate embodiment of the present invention.

Methods for preparing peptide vaccines are well known in the art and are described, for example, in EP1408048, United States Patent Application Number 20070154953, and OGASAWARA et al (Proc. Nati. Acad. Sci. USA Vol. 89, pp. 8995-8999, October 1992). In one embodiment, peptide evolution techniques are used to create an antigen with higher immunogenicity. Techniques for peptide evolution are well known in the art and are described, for example in U.S. Pat. No. 6,773,900.

In one embodiment, a vaccine is a composition which elicits an immune response to an antigen or polypeptide in the composition as a result of exposure to the composition.

In another embodiment, the present invention provides a recombinant Listeria strain comprising a recombinant nucleotide molecule of the present invention.

The recombinant Listeria strain of methods and compositions of the present invention is, in another embodiment, a recombinant Listeria monocytogenes strain. In another embodiment, the Listeria strain is a recombinant Listeria seeligeri strain. In another embodiment, the Listeria strain is a recombinant Listeria grayi strain. In another embodiment, the Listeria strain is a recombinant Listeria ivanovii strain. In another embodiment, the Listeria strain is a recombinant Listeria murrayi strain. In another embodiment, the Listeria strain is a recombinant Listeria welshimeri strain. In another embodiment, the Listeria strain is a recombinant strain of any other Listeria species known in the art. In one embodiment, the Listeria strain is a Listeria strain comprising LLO, while in another embodiment, the Listeria strain is a Listeria strain comprising ActA, while in another embodiment, the Listeria strain is a Listeria strain comprising PEST-like sequences.

In another embodiment the Listeria strain is attenuated by deletion of a gene. In another embodiment the Listeria strain is attenuated by deletion of more than 1 gene. In another embodiment the Listeria strain is attenuated by deletion or inactivation of a gene. In another embodiment the Listeria strain is attenuated by deletion or inactivation of more than 1 gene.

In another embodiment, the gene that is mutated is hly. In another embodiment, the gene that is mutated is actA. In another embodiment, the gene that is mutated is plc A. In another embodiment, the gene that is mutated is plcB. In another embodiment, the gene that is mutated is mpl. In another embodiment, the gene that is mutated is inl A. In another embodiment, the gene that is mutated is inlB. In another embodiment, the gene that is mutated is bsh.

In another embodiment, the Listeria strain is an auxotrophic mutant. In another embodiment, the Listeria strain is deficient in a gene encoding a vitamin synthesis gene. In another embodiment, the Listeria strain is deficient in a gene encoding pantothenic acid synthase.

In another embodiment, the Listeria strain is deficient in an AA metabolism enzyme. In another embodiment the Listeria strain is deficient in a D-glutamic acid synthase gene. In another embodiment the Listeria strain is deficient in the dat gene. In another embodiment the Listeria strain is deficient in the dal gene. In another embodiment the Listeria strain is deficient in the dga gene. In another embodiment the Listeria strain is deficient in a gene involved in the synthesis of diaminopimelic acid. CysK. In another embodiment, the gene is vitamin-B12 independent methionine synthase. In another embodiment, the gene is trpA. In another embodiment, the gene is trpB. In another embodiment, the gene is trpE. In another embodiment, the gene is asnB. In another embodiment, the gene is gltD. In another embodiment, the gene is gltB. In another embodiment, the gene is leuA. In another embodiment, the gene is argG. In another embodiment, the gene is thrC. In another embodiment, the Listeria strain is deficient in one or more of the genes described hereinabove.

In another embodiment, the Listeria strain is deficient in a synthase gene. In another embodiment, the gene is an AA synthesis gene. In another embodiment, the gene is folP. In another embodiment, the gene is dihydrouridine synthase family protein. In another embodiment, the gene is ispD. In another embodiment, the gene is ispF. In another embodiment, the gene is phosphoenolpyruvate synthase. In another embodiment, the gene is hisF. In another embodiment, the gene is hisH. In another embodiment, the gene is fliI. In another embodiment, the gene is ribosomal large subunit pseudouridine synthase. In another embodiment, the gene is ispD. In another embodiment, the gene is bifunctional GMP synthase/glutamine amidotransferase protein. In another embodiment, the gene is cobS. In another embodiment, the gene is cobB. In another embodiment, the gene is cbiD. In another embodiment, the gene is uroporphyrin-III C-methyltransferase/uroporphyrinogen-III synthase. In another embodiment, the gene is cobQ. In another embodiment, the gene is uppS. In another embodiment, the gene is truB. In another embodiment, the gene is dxs. In another embodiment, the gene is mvaS. In another embodiment, the gene is dapA. In another embodiment, the gene is ispG. In another embodiment, the gene is folC. In another embodiment, the gene is citrate synthase. In another embodiment, the gene is argJ. In another embodiment, the gene is 3-deoxy-7-phosphoheptulonate synthase. In another embodiment, the gene is indole-3-glycerol-phosphate synthase. In another embodiment, the gene is anthranilate synthase/glutamine amidotransferase component. In another embodiment, the gene is menB. In another embodiment, the gene is menaquinone-specific isochorismate synthase. In another embodiment, the gene is phosphoribosylformylglycinamidine synthase I or II. In another embodiment, the gene is phosphoribosylaminoimidazole-succinocarboxamide synthase. In another embodiment, the gene is carB. In another embodiment, the gene is carA. In another embodiment, the gene is thyA. In another embodiment, the gene is mgsA. In another embodiment, the gene is aroB. In another embodiment, the gene is hepB. In another embodiment, the gene is rluB. In another embodiment, the gene is ilvB. In another embodiment, the gene is ilvN. In another embodiment, the gene is alsS. In another embodiment, the gene is fabF. In another embodiment, the gene is fabH. In another embodiment, the gene is pseudouridine synthase. In another embodiment, the gene is pyrG. In another embodiment, the gene is truA. In another embodiment, the gene is pabB. In another embodiment, the gene is an atp synthase gene (e.g. atpC, atpD-2, aptG, atpA-2, etc).

In another embodiment, the gene is phoP. In another embodiment, the gene is aroA aroC. In another embodiment, the gene is aroD. In another embodiment, the gene is plcB.

In another embodiment, the Listeria strain is deficient in a peptide transporter. In another embodiment, the gene is ABC transporter/ATP-binding/permease protein. In another embodiment, the gene is oligopeptide ABC transporter/oligopeptide-binding protein. In another embodiment, the gene is oligopeptide ABC transporter/permease protein. In another embodiment, the gene is zinc ABC transporter/zinc-binding protein. In another embodiment, the gene is sugar ABC transporter. In another embodiment, the gene is phosphate transporter. In another embodiment, the gene is ZIP zinc transporter. In another embodiment, the gene is drug resistance transporter of the EmrB/QacA family. In another embodiment, the gene is sulfate transporter. In another embodiment, the gene is proton-dependent oligopeptide transporter. In another embodiment, the gene is magnesium transporter. In another embodiment, the gene is formate/nitrite transporter. In another embodiment, the gene is spermidine/putrescine ABC transporter. In another embodiment, the gene is Na/Pi-cotransporter. In another embodiment, the gene is sugar phosphate transporter. In another embodiment, the gene is glutamine ABC transporter. In another embodiment, the gene is major facilitator family transporter. In another embodiment, the gene is glycine betaine/L-proline ABC transporter. In another embodiment, the gene is molybdenum ABC transporter. In another embodiment, the gene is techoic acid ABC transporter. In another embodiment, the gene is cobalt ABC transporter. In another embodiment, the gene is ammonium transporter. In another embodiment, the gene is amino acid ABC transporter. In another embodiment, the gene is cell division ABC transporter. In another embodiment, the gene is manganese ABC transporter. In another embodiment, the gene is iron compound ABC transporter. In another embodiment, the gene is maltose/maltodextrin ABC transporter. In another embodiment, the gene is drug resistance transporter of the Bcr/CflA family.

In another embodiment, the gene is a subunit of one of the above proteins.

In another embodiment, a recombinant Listeria strain of the present invention has been passaged through an animal host. In another embodiment, the passaging maximizes efficacy of the strain as a vaccine vector. In another embodiment, the passaging stabilizes the immunogenicity of the Listeria strain. In another embodiment, the passaging stabilizes the virulence of the Listeria strain. In another embodiment, the passaging increases the immunogenicity of the Listeria strain. In another embodiment, the passaging increases the virulence of the Listeria strain. In another embodiment, the passaging removes unstable sub-strains of the Listeria strain. In another embodiment, the passaging reduces the prevalence of unstable sub-strains of the Listeria strain. In another embodiment, the passaging attenuates the strain, or in another embodiment, makes the strain less virulent. Methods for passaging a recombinant Listeria strain through an animal host are well known in the art, and are described, for example, in U.S. patent application Ser. No. 10/541,614. Each possibility represents a separate embodiment of the present invention.

Each Listeria strain and type thereof represents a separate embodiment of the present invention.

In another embodiment, the recombinant Listeria of methods and compositions of the present invention is stably transformed with a construct encoding an antigen or an LLO-antigen fusion. In one embodiment, the construct contains a polylinker to facilitate further subcloning. Several techniques for producing recombinant Listeria are known; each technique represents a separate embodiment of the present invention.

In another embodiment, the construct or heterologous gene is integrated into the Listerial chromosome using homologous recombination. Techniques for homologous recombination are well known in the art, and are described, for example, in Frankel, F R, Hegde, S, Lieberman, J, and Y Paterson. Induction of a cell-mediated immune response to HIV gag using Listeria monocytogenes as a live vaccine vector. J. Immunol. 155: 4766-4774. 1995; Mata, M, Yao, Z, Zubair, A, Syres, K and Y Paterson, Evaluation of a recombinant Listeria monocytogenes expressing an HIV protein that protects mice against viral challenge. Vaccine 19:1435-45, 2001; Boyer, J D, Robinson, T M, Maciag, P C, Peng, X, Johnson, R S, Paviakis, G, Lewis, M G, Shen, A, Siliciano, R, Brown, C R, Weiner, D, and Y Paterson. DNA prime Listeria boost induces a cellular immune response to SIV antigens in the Rhesus Macaque model that is capable of limited suppression of SIV239 viral replication. Virology. 333: 88-101, 2005. In another embodiment, homologous recombination is performed as described in U.S. Pat. No. 6,855,320. In another embodiment, a temperature sensitive plasmid is used to select the recombinants. Each technique represents a separate embodiment of the present invention.

In another embodiment, the construct or heterologous gene is integrated into the Listerial chromosome using transposon insertion. Techniques for transposon insertion are well known in the art, and are described, inter alia, by Sun et al. (Infection and Immunity 1990, 58: 3770-3778) in the construction of DP-L967. Transposon mutagenesis has the advantage, in another embodiment, that a stable genomic insertion mutant can be formed. In another embodiment, the position in the genome where the foreign gene has been inserted by transposon mutagenesis is unknown.

In another embodiment, the construct or heterologous gene is integrated into the Listerial chromosome using phage integration sites (Lauer P, Chow M Y et al, Construction, characterization, and use of two LM site-specific phage integration vectors. J Bacteriol 2002; 184(15): 4177-86). In another embodiment, an integrase gene and attachment site of a bacteriophage (e.g. U153 or PSA listeriophage) is used to insert the heterologous gene into the corresponding attachment site, which can be any appropriate site in the genome (e.g. comK or the 3′ end of the arg tRNA gene). In another embodiment, endogenous prophages are cured from the attachment site utilized prior to integration of the construct or heterologous gene. In another embodiment, this method results in single-copy integrants. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the construct is carried by the Listeria strain on a plasmid. LM vectors that express antigen fusion proteins have been constructed via this technique. Lm-GG/E7 was made by complementing a prfA-deletion mutant with a plasmid containing a copy of the prfA gene and a copy of the E7 gene fused to a form of the LLO (hly) gene truncated to eliminate the hemolytic activity of the enzyme, as described herein. Functional LLO was maintained by the organism via the endogenous chromosomal copy of hly. In another embodiment, the plasmid contains an antibiotic resistance gene. In another embodiment, the plasmid contains a gene encoding a virulence factor that is lacking in the genome of the transformed Listeria strain. In another embodiment, the virulence factor is prfA. In another embodiment, the virulence factor is LLO. In another embodiment, the virulence factor is ActA. In another embodiment, the virulence factor is any of the genes enumerated above as targets for attenuation. In another embodiment, the virulence factor is any other virulence factor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, a recombinant peptide of the present invention is fused to a Listerial protein, such as PI-PLC, or a construct encoding same. In another embodiment, a signal sequence of a secreted Listerial protein such as hemolysin, ActA, or phospholipases is fused to the antigen-encoding gene. In another embodiment, a signal sequence of the recombinant vaccine vector is used. In another embodiment, a signal sequence functional in the recombinant vaccine vector is used. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the construct is contained in the Listeria strain in an episomal fashion. In another embodiment, the foreign antigen is expressed from a vector harbored by the recombinant Listeria strain.

Each method of expression in Listeria represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of inducing an anti-HMW-MAA immune response in a subject, comprising administering to the subject a composition comprising a recombinant Listeria strain of the present invention, thereby inducing an anti-HMW-MAA immune response in a subject.

In another embodiment, a subject is administered his/her own allogeneic cells, which in one embodiment, elicit an immune response to an antigen. In another embodiment, the compositions and methods of the present invention result in the expression of stimulatory cytokines, which in one embodiment, are Th1 cytokines, which in one embodiment, is IFN-gamma. In one embodiment, the expression of stimulatory cytokines contributes to the anti-tumor effect of the compositions and methods. In another embodiment, the compositions and methods of the present invention result in the expression of gamma delta T cells.

In another embodiment, the present invention provides compositions and methods for inducing non-specific anti-tumor responses. In one embodiment, immunization with a melanoma antigen, such as HMW-MAA peptide, protects against a type of melanoma that does not express the antigen (Example 4).

In another embodiment, the present invention provides a method of inducing an immune response against an HMW-MAA-expressing tumor in a subject, comprising administering to the subject a composition comprising a recombinant Listeria strain of the present invention, thereby inducing an immune response against an HMW-MAA-expressing tumor. As provided herein, vaccines of the present invention induce antigen-specific immune response, as shown by multiple lines of evidence—e.g. inhibition of tumor growth, tetramer staining, measurement of numbers of tumor-infiltrating CD8⁺ T cells, FACS, and chromium release assay.

In another embodiment, the present invention provides a method of inducing an immune response against a pericyte in a subject, comprising administering to the subject a composition comprising a recombinant Listeria strain of the present invention, thereby inducing an immune response against a pericyte.

In another embodiment, the present invention provides a method of impeding the growth of a solid tumor in a subject, comprising administering to the subject a composition comprising a recombinant Listeria strain of the present invention, thereby impeding a growth and/or delaying progression of a solid tumor in a subject. In another embodiment, the subject mounts an immune response against a pericyte of the solid tumor. In another embodiment, the pericyte is in a vasculature of the solid tumor. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of treating a solid tumor in a subject, comprising administering to the subject a composition comprising a recombinant Listeria strain of the present invention, thereby treating a solid tumor in a subject. In another embodiment, the subject mounts an immune response against a pericyte of the solid tumor. In another embodiment, the pericyte is in a vasculature of the solid tumor. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of lysing one or more tumor cells in a subject, comprising administering to the subject a composition comprising a recombinant Listeria strain of the present invention, thereby lysing one or more tumor cells in a subject. In one embodiment, tumor lysis is due to cytotoxic T lymphocytes, tumor infilitrating lymphocytes, or a combination thereof, which in one embodiment are tumor-specific.

In one embodiment, methods of the present invention are used to treat, impede, suppress, inhibit, or prevent any of the above-described diseases, disorders, symptoms, or side effects associated with allergy or asthma. In one embodiment, “treating” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or lessen the targeted pathologic condition or disorder as described hereinabove. Thus, in one embodiment, treating may include directly affecting or curing, suppressing, inhibiting, preventing, reducing the severity of, delaying the onset of, reducing symptoms associated with the disease, disorder or condition, or a combination thereof. Thus, in one embodiment, “treating” refers inter alia to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of or decreasing resistance to alternative therapeutics, or a combination thereof. In one embodiment, “preventing” or “impeding” refers, inter alia, to delaying the onset of symptoms, preventing relapse to a disease, decreasing the number or frequency of relapse episodes, increasing latency between symptomatic episodes, or a combination thereof. In one embodiment, “suppressing” or “inhibiting”, refers inter alia to reducing the severity of symptoms, reducing the severity of an acute episode, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, or a combination thereof.

In one embodiment, symptoms are primary, while in another embodiment, symptoms are secondary. In one embodiment, “primary” refers to a symptom that is a direct result of a particular disease or disorder, while in one embodiment, “secondary” refers to a symptom that is derived from or consequent to a primary cause. In one embodiment, the compounds for use in the present invention treat primary or secondary symptoms or secondary complications related to allergy or asthma. In another embodiment, “symptoms” may be any manifestation of a disease or pathological condition.

As provided herein, Listeria strains expressing HMW-MAA inhibited growth of tumors that did not express HMW-MAA. These findings show that anti-HMW-MAA immune responses inhibit and reverse vascularization of, and thus inhibit growth of, solid tumors. Anti-HMW-MAA vaccines of the present invention were able to exert these effects in spite of the incomplete identity (80%) between HMW-MAA and its mouse homolog, nameely mouse chondroitin sulfate proteoglycan (“AN2”). In this embodiment, anti-HMW-MAA vaccines of the present invention are efficacious for vaccination against any solid tumor, regardless of its expression of HMW-MAA. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of impeding a vascularization of a solid tumor in a subject, comprising administering to the subject a composition comprising a recombinant Listeria strain of the present invention, thereby impeding a vascularization of a solid tumor in a subject. In another embodiment, the subject mounts an immune response against a pericyte of the solid tumor. In another embodiment, the pericyte is in a vasculature of the solid tumor. Each possibility represents a separate embodiment of the present invention.

The solid tumor that is the target of methods and compositions of the present invention is, in another embodiment, a melanoma. In another embodiment, the tumor is a sarcoma. In another embodiment, the tumor is a carcinoma. In another embodiment, the tumor is a mesothelioma (e.g. malignant mesothelioma). In another embodiment, the tumor is a glioma. In another embodiment, the tumor is a germ cell tumor. In another embodiment, the tumor is a choriocarcinoma.

In another embodiment, the tumor is pancreatic cancer. In another embodiment, the tumor is ovarian cancer. In another embodiment, the tumor is gastric cancer. In another embodiment, the tumor is a carcinomatous lesion of the pancreas. In another embodiment, the tumor is pulmonary adenocarcinoma. In another embodiment, the tumor is colorectal adenocarcinoma. In another embodiment, the tumor is pulmonary squamous adenocarcinoma. In another embodiment, the tumor is gastric adenocarcinoma. In another embodiment, the tumor is an ovarian surface epithelial neoplasm (e.g. a benign, proliferative or malignant variety thereof). In another embodiment, the tumor is an oral squamous cell carcinoma. In another embodiment, the tumor is non small-cell lung carcinoma. In another embodiment, the tumor is an endometrial carcinoma. In another embodiment, the tumor is a bladder cancer. In another embodiment, the tumor is a head and neck cancer. In another embodiment, the tumor is a prostate carcinoma.

In another embodiment, the tumor is a non-small cell lung cancer (NSCLC). In another embodiment, the tumor is a Wilms' tumor. In another embodiment, the tumor is a desmoplastic small round cell tumor. In another embodiment, the tumor is a colon cancer. In another embodiment, the tumor is a lung cancer. In another embodiment, the tumor is an ovarian cancer. In another embodiment, the tumor is a uterine cancer. In another embodiment, the tumor is a thyroid cancer. In another embodiment, the tumor is a hepatocellular carcinoma. In another embodiment, the tumor is a thyroid cancer. In another embodiment, the tumor is a liver cancer. In another embodiment, the tumor is a renal cancer. In another embodiment, the tumor is a kaposis. In another embodiment, the tumor is a sarcoma. In another embodiment, the tumor is another carcinoma or sarcoma. Each possibility represents a separate embodiment of the present invention.

In one embodiment, this invention provides compositions and methods for preventing cancer in populations that are predisposed to the cancer or in populations that are at high risk for the cancer, which in one embodiment, may be a population of women with brcal or 2 mutations, which population in one embodiment is susceptible to breast cancer.

Each of the above types of cancer represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of inducing an immune response against an HMW-MAA-expressing tumor in a subject, comprising administering to the subject a composition comprising a recombinant Listeria strain of the present invention, thereby inducing an immune response against an HMW-MAA-expressing tumor. As provided herein, Listeria strains expressing HMW-MAA elicited anti-HMW-MAA immune responses and inhibited growth HMW-MAA-expressing of tumors.

The HMW-MAA-expressing tumor that is the target of methods and compositions of the present invention is, in another embodiment, a basal cell carcinoma. In another embodiment, the HMW-MAA-expressing tumor is a tumor of neural crest origin. In another embodiment, the HMW-MAA-expressing tumor is an astrocytoma. In another embodiment, the HMW-MAA-expressing tumor is a glioma. In another embodiment, the HMW-MAA-expressing tumor is a neuroblastoma. In another embodiment, the HMW-MAA-expressing tumor is a sarcoma. In another embodiment, the HMW-MAA-expressing tumor is a childhood leukemia. In another embodiment, the HMW-MAA-expressing tumor is a lobular breast carcinoma lesion. In another embodiment, the HMW-MAA-expressing tumor is a melanoma. In another embodiment, the HMW-MAA-expressing tumor is any other HMW-MAA-expressing tumor. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method ofinducingan immuneresponse against a pericyte in a subject, comprising administering to the subject a composition comprising a recombinant Listeria strain of the present invention, thereby inducing an immune response against a pericyte. As provided herein, Listeria strains expressing HMW-MAA inhibited growth of solid tumors, even those that did not express HMW-MAA. These findings demonstrate inhibition of vascularization via induction of immune responses against tumor-vascular associated pericytes.

In another embodiment, the present invention provides a method of impeding a growth and/or delaying progression of a HMW-MAA-expressing tumor in a subject, comprising administering to the subject a composition comprising a recombinant Listeria strain of the present invention, thereby impeding a growth and/or delaying progression of a HMW-MAA-expressing tumor in a subject. In another embodiment, the subject mounts an immune response against the HMW-MAA-expressing tumor. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of treating a HMW-MAA-expressing tumor in a subject, comprising administering to the subject a composition comprising a recombinant Listeria strain of the present invention, thereby treating a HMW-MAA-expressing tumor in a subject. In another embodiment, the subject mounts an immune response against the HMW-MAA-expressing tumor. Each possibility represents a separate embodiment of the present invention.

The recombinant Listeria strain of the present invention utilized in methods of the present invention comprises, in another embodiment, a recombinant polypeptide of the present invention. In another embodiment, the recombinant Listeria strain comprises a recombinant nucleotide molecule of the present invention. In another embodiment, the recombinant Listeria strain is any recombinant Listeria strain described or enumerated above. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of inducing an anti-HMW-MAA immune response in a subject, comprising administering to the subject a composition comprising a recombinant polypeptide of the present invention, thereby inducing an anti-HMW-MAA immune response in a subject.

In another embodiment, the present invention provides a method of inducing an immune response against an HMW-MAA-expressing tumor in a subject, comprising administering to the subject a composition comprising a recombinant polypeptide of the present invention, thereby inducing an immune response against an HMW-MAA-expressing tumor.

In another embodiment, the present invention provides a method of inducing an immune response against a pericyte in a subject, comprising administering to the subject a composition comprising a recombinant polypeptide of the present invention, thereby inducing an immune response against a pericyte.

In another embodiment, the present invention provides a method of impeding a growth and/or delaying progression of a solid tumor in a subject, comprising administering to the subject a composition comprising a recombinant polypeptide of the present invention, thereby impeding a growth and/or delaying progression of a solid tumor in a subject. In another embodiment, the subject mounts an immune response against a pericyte of the solid tumor. In another embodiment, the pericyte is in a vasculature of the solid tumor. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of treating a solid tumor in a subject, comprising administering to the subject a composition comprising a recombinant polypeptide of the present invention, thereby treating a solid tumor in a subject. In another embodiment, the subject mounts an immune response against a pericyte of the solid tumor. In another embodiment, the pericyte is in a vasculature of the solid tumor. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of impeding a vascularization of a solid tumor in a subject, comprising administering to the subject a composition comprising a recombinant polypeptide of the present invention, thereby impeding a vascularization of a solid tumor in a subject. In another embodiment, the subject mounts an immune response against a pericyte of the solid tumor. In another embodiment, the pericyte is in a vasculature of the solid tumor. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of impeding a growth and/or delaying progression of a HMW-MAA-expressing tumor in a subject, comprising administering to the subject a composition comprising a recombinant polypeptide of the present invention, thereby impeding a growth and/or delaying progression of a HMW-MAA-expressing tumor in a subject. In another embodiment, the subject mounts an immune response against the HMW-MAA-expressing tumor. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of treating a HMW-MAA-expressing tumor in a subject, comprising administering to the subject a composition comprising a recombinant polypeptide of the present invention, thereby treating a HMW-MAA-expressing tumor in a subject. In another embodiment, the subject mounts an immune response against the HMW-MAA-expressing tumor. Each possibility represents a separate embodiment of the present invention.

In other embodiments, the recombinant polypeptide of any of the methods described above have any of the characteristics of a recombinant polypeptide of compositions of the present invention. Each characteristic represents a separate embodiment of the present invention.

In another embodiment of methods of the present invention, a vaccine comprising a recombinant Listeria strain of the present invention is administered. In another embodiment, an immunogenic composition comprising a recombinant Listeria strain of the present invention is administered. In another embodiment, a vaccine comprising a recombinant polypeptide of the present invention is administered. In another embodiment, an immunogenic composition comprising a recombinant polypeptide of the present invention is administered. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the target pericyte of methods and compositions of the present invention is an activated pericyte. In another embodiment, the target pericyte is any other type of pericyte known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, a method or immunogenic composition of methods and compositions of the present invention induces a cell-mediated immune response. In another embodiment, the immunogenic composition induces a predominantly cell-mediated immune response. In another embodiment, the immunogenic composition induces a predominantly Th1-type immune response. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the immune response elicited by methods of the present invention is a cell-mediated immune response. In another embodiment, the immune response is a T-cell-mediated immune response. Each possibility represents a separate embodiment of the present invention.

The T cell-mediated immune response induced by methods and compositions of the present invention comprises, in another embodiment, a CTL. In another embodiment, the T cell involved in the T cell-mediated immune response is a CTL. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the T cell-mediated immune response comprises a T helper cell. In another embodiment, the T cell involved in the T cell-mediated immune response is a T helper cell. Each possibility represents a separate embodiment of the present invention.

In another embodiment of methods of the present invention, the subject is immunized with an immunogenic composition, vector, or recombinant peptide of the present invention. In another embodiment, the subject is contacted with the immunogenic composition, vector, or recombinant peptide. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of inhibiting adhesion of a cancer cell to the extracellular matrix, comprising inducing an anti-HMW-MAA immune response by a method of the present invention, thereby inhibiting adhesion of a cancer cell to the extracellular matrix. In another embodiment, the cancer cell is a melanoma cell. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of inhibiting metastasis of a tumor, comprising inducing an anti-HMW-MAA immune response by a method of the present invention, thereby inhibiting metastasis of a tumor. In another embodiment, the tumor is a melanoma tumor. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of inhibiting migration of a cancer cell, comprising inducing an anti-HMW-MAA immune response by a method of the present invention, thereby inhibiting migration of a cancer cell. In another embodiment, the cancer cell is a melanoma cell. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of inhibiting proliferation of cells in a tumor, comprising inducing an anti-HMW-MAA immune response by a method of the present invention, thereby inhibiting proliferation of cells in a tumor. In another embodiment, the tumor is a melanoma tumor. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of reducing invasiveness of a tumor, comprising inducing an anti-HMW-MAA immune response by a method of the present invention, thereby reducing invasiveness of a tumor. In another embodiment, the tumor is a melanoma tumor. In another embodiment, anti-HMW-MAA immune responses inhibit formation of HMW-MAA-MT3-MMP (membrane type metalloproteinases) complexes. In another embodiment, inhibition of formation of these complexes inhibits degradation of type I collagen by melanoma cells. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of inhibiting conversion of plasminogen into plasmin in the vicinity of a tumor, comprising inducing an anti-HMW-MAA immune response by a method of the present invention, thereby inhibiting conversion of plasminogen into plasmin in the vicinity of a tumor. In another embodiment, the tumor is a melanoma tumor. In another embodiment, inhibiting plasmin release inhibits, in turn, degradation of the extracellular matrix (ECM). Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of inhibiting sequestration of angiostatin in the vicinity of a tumor, comprising inducing an anti-HMW-MAA immune response by a method of the present invention, thereby inhibiting sequestration of angiostatin in the vicinity of a tumor. In another embodiment, the tumor is a melanoma tumor. Each possibility represents a separate embodiment of the present invention.

In another embodiment, a peptide of the present invention is homologous to a peptide enumerated herein. The terms “homology,” “homologous,” etc, when in reference to any protein or peptide, refer, in one embodiment, to a percentage of amino acid residues in the candidate sequence that are identical with the residues of a corresponding native polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Methods and computer programs for the alignment are well known in the art.

Homology is, in another embodiment, determined by computer algorithm for sequence alignment, by methods well described in the art. For example, computer algorithm analysis of nucleic acid sequence homology can include the utilization of any number of software packages available, such as, for example, the BLAST, DOMAIN, BEAUTY (BLAST Enhanced Alignment Utility), GENPEPT and TREMBL packages.

In another embodiment, “homology” or “homologous” refers to identity to a non-HMW-MAA sequence selected from SEQ ID No: 1-14 of greater than 70%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 1-14 of greater than 72%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-14 of greater than 75%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 1-14 of greater than 78%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-14 of greater than 80%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-14 of greater than 82%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 1-14 of greater than 83%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-14 of greater than 85%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-14 of greater than 87%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 1-14 of greater than 88%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-14 of greater than 90%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-14 of greater than 92%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 1-14 of greater than 93%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-14 of greater than 95%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 1-14 of greater than 96%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-14 of greater than 97%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-14 of greater than 98%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-14 of greater than 99%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-14 of 100%. Each possibility represents a separate embodiment of the present invention.

In another embodiment, “homology” or “homologous” refers to identity to an HMW-MAA sequence selected from SEQ ID No: 15-16 of greater than 70%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 15-16 of greater than 72%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 75%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 15-16 of greater than 78%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 80%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 82%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 15-16 of greater than 83%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 85%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 87%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 15-16 of greater than 88%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 90%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 92%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 15-16 of greater than 93%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 95%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 15-16 of greater than 96%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 97%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 98%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 15-16 of greater than 99%. In another embodiment; “homology” refers to identity to one of SEQ ID No: 15-16 of 100%. Each possibility represents a separate embodiment of the present invention.

In another embodiment, homology is determined via determination of candidate sequence hybridization, methods of which are well described in the art (See, for example, “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., Eds. (1985); Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y). In other embodiments, methods of hybridization are carried out under moderate to stringent conditions, to the complement of a DNA encoding a native caspase peptide. Hybridization conditions being, for example, overnight incubation at 42° C. in a solution comprising: 10-20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA.

Protein and/or peptide homology for any AA sequence listed herein is determined, in another embodiment, by methods well described in the art, including immunoblot analysis, or via computer algorithm analysis of AA sequences, utilizing any of a number of software packages available, via established methods. Some of these packages include the FASTA, BLAST, MPsrch or Scanps packages, and, in another embodiment, employ the use of the Smith and Waterman algorithms, and/or globalaocal or BLOCKS alignments for analysis. Each method of determining homology represents a separate embodiment of the present invention.

In another embodiment of the present invention, “nucleic acids” or “nucleotide” refers to a string of at least two base-sugar-phosphate combinations. The term includes, in one embodiment, DNA and RNA. “Nucleotides” refers, in one embodiment, to the monomeric units of nucleic acid polymers. RNA is, in one embodiment, in the form of a tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, small inhibitory RNA (siRNA), micro RNA (miRNA) and ribozymes. The use of siRNA and miRNA has been described (Caudy A A et al, Genes & Devel 16: 2491-96 and references cited therein). DNA can be, in other embodiments, in form of plasmid DNA, viral DNA, linear DNA, or chromosomal DNA or derivatives of these groups. In addition, these forms of DNA and RNA can be single, double, triple, or quadruple stranded. The term also includes, in another embodiment, artificial nucleic acids that contain other types of backbones but the same bases. In one embodiment, the artificial nucleic acid is a PNA (peptide nucleic acid). PNA contain peptide backbones and nucleotide bases and are able to bind, in one embodiment, to both DNA and RNA molecules. In another embodiment, the nucleotide is oxetane modified. In another embodiment, the nucleotide is modified by replacement of one or more phosphodiester bonds with a phosphorothioate bond. In another embodiment, the artificial nucleic acid contains any other variant of the phosphate backbone of native nucleic acids known in the art. The use of phosphothiorate nucleic acids and PNA are known to those skilled in the art, and are described in, for example, Neilsen P E, Curr Opin Struct Biol 9:353-57; and Raz N K et al Biochem Biophys Res Commun. 297:1075-84. The production and use of nucleic acids is known to those skilled in art and is described, for example, in Molecular Cloning, (2001), Sambrook and Russell, eds. and Methods in Enzymology: Methods for molecular cloning in eukaryotic cells (2003) Purchio and G. C. Fareed. Each nucleic acid derivative represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a kit comprising a compound or composition utilized in performing a method of the present invention. In another embodiment, the present invention provides a kit comprising a composition, tool, or instrument of the present invention. Each possibility represents a separate embodiment of the present invention.

Pharmaceutical Compositions and Methods of Administration

“Pharmaceutical composition” refers, in another embodiment, to a therapeutically effective amount of the active ingredient, i.e. the recombinant peptide or vector comprising or encoding same, together with a pharmaceutically acceptable carrier or diluent. A “therapeutically effective amount” refers, in another embodiment, to that amount which provides a therapeutic effect for a given condition and administration regimen.

The pharmaceutical compositions containing the active ingredient can be, in another embodiment, administered to a subject by any method known to a person skilled in the art, such as parenterally, transmucosally, transdermally, intramuscularly, intravenously, intra-dermally, subcutaneously, intra-peritonealy, intra-ventricularly, intra-cranially, intra-vaginally, or intra-tumorally.

In another embodiment of methods and compositions of the present invention, the pharmaceutical compositions are administered orally, and are thus formulated in a form suitable for oral administration, i.e. as a solid or a liquid preparation. Suitable solid oral formulations include tablets, capsules, pills, granules, pellets and the like. Suitable liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In another embodiment of the present invention, the active ingredient is formulated in a capsule. In accordance with this embodiment, the compositions of the present invention comprise, in addition to the active compound and the inert carrier or diluent, a hard gelating capsule.

In another embodiment, the pharmaceutical compositions are administered by intravenous, intra-arterial, or intra-muscular injection of a liquid preparation. Suitable liquid formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In another embodiment, the pharmaceutical compositions are administered intravenously and are thus formulated in a form suitable for intravenous administration. In another embodiment, the pharmaceutical compositions are administered intra-arterially and are thus formulated in a form suitable for intra-arterial administration. In another embodiment, the pharmaceutical compositions are administered intra-muscularly and are thus formulated in a form suitable for intra-muscular administration.

In another embodiment, the pharmaceutical compositions are administered topically to body surfaces and are thus formulated in a form suitable for topical administration. Suitable topical formulations include gels, ointments, creams, lotions, drops and the like. For topical administration, the recombinant peptide or vector is prepared and applied as a solution, suspension, or emulsion in a physiologically acceptable diluent with or without a pharmaceutical carrier.

In another embodiment, the active ingredient is delivered in a vesicle, e.g. a liposome.

In other embodiments, carriers or diluents used in methods of the present invention include, but are not limited to, a gum, a starch (e.g. corn starch, pregeletanized starch), a sugar (e.g., lactose, mannitol, sucrose, dextrose), a cellulosic material (e.g. microcrystalline cellulose), an acrylate (e.g. polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.

In other embodiments, pharmaceutically acceptable carriers for liquid formulations are aqueous or non-aqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipid from milk or eggs.

In another embodiment, parenteral vehicles (for subcutaneous, intravenous, intraarterial, or intramuscular injection) include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. Examples of oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipid from milk or eggs.

In another embodiment, the pharmaceutical compositions provided herein are controlled-release compositions, i.e. compositions in which the active ingredient is released over a period of time after administration. Controlled- or sustained-release compositions include formulation in lipophilic depots (e.g. fatty acids, waxes, oils). In another embodiment, the composition is an immediate-release composition, i.e. a composition in which all the active ingredient is released immediately after administration.

EXPERIMENTAL DETAILS SECTION Example 1 Construction of LLO-HMW-MAA Constructs and Listeria Strains Expressing Same

LLO-HMW-MAA constructs were created as follows:

pGG-55, the precursor of the LLO-HMW-MAA constructs, was created from pAM401, a shuttle vector able to replicate in both gram-negative and gram-positive bacteria (Wirth R et al, J Bacteriol, 165: 831, 1986). pAM401 contains a gram-positive chloramphenicol resistance gene and gram negative tetracycline resistance determinant. In pGG-55, the hly promoter drives the expression of the first 441 AA of the hly gene product, (lacking the hemolytic C-terminus, having the sequence set forth in SEQ ID No: 3), which is joined by the XhoI site to the E7 gene, yielding a hly-E7 fusion gene that is transcribed and secreted as LLO-E7.

Generation of pGG-55: A fusion of a listeriolysin fragment to E7 (“LLO-E7”) and the pluripotential transcription factor prfA were subcloned in pAM401 as follows: The DNA fragment encoding the first 420 AA of LLO and its promoter and upstream regulatory sequences was PCR amplified with LM genomic DNA used as a template and ligated into pUC19. PCR primers used were 5′-GGCCCGGGCCCCCTCCTTTGAT-3′ (SEQ ID No: 17) and 5′-GGTCTAGATCATAATTTACTTCATCC-3′ (SEQ ID No: 18). E7 was amplified by PCR using the primers 5′-GGCTCGAGCATGGAGATACACC-3′ (SEQ ID No: 19; XhoI site is underlined) and 5′-GGGGACTAGTTTATGGTTTCTGAGAACA-3′ (SEQ ID No: 20; SpeI site is underlined) and ligated into pCR2.1 (Invitrogen, San Diego, Calif.). E7 was excised from pCR2.1 by Xhol/SpeI digestion and subsequently ligated as an in-frame translational fusion into pUC19-hly downstream of the hemolysin gene fragment. The fusion was then subcloned into the multilinker of pAM401. The prfA gene was then subcloned into the SalI site of the resulting plasmid, yielding pGG-55 (FIG. 1).

pGG34A, B and C were created from pGG-55 as follows:

HMW-MAA fragments A, B, and C (encoding AA 360-554, 701-1130, and 2160-2258, respectively, FIG. 1) have the following sequences:

HMW-MAA-A: (SEQ ID No: 21) Ttcaatggccagaggcgggggctgcgggaagctttgctgacgcgcaacatggcagccggctgcaggctggaggaggaggagtatgag gacgatgcctatggacattatgaagctttctccaccctggcccctgaggcttggccagccatggagctgcctgagccatgcgtgcctgagccagggctg cctcctgtctttgccaatttcacccagctgctgactatcagcccactggtggtggccgaggggggcacagcctggcttgagtggaggcatgtgcagccc acgctggacctgatggaggctgagctgcgcaaatcccaggtgctgttcagcgtgacccgaggggcacgccatggcgagctcgagctggacatcccg ggagcccaggcacgaaaaatgttcaccctcctggacgtggtgaaccgcaaggcccgcttcatccacgatggctctgaggacacctccgaccagctggt gctggaggtgtcggtgacggctcgggtgcccatgccctcatgccttcggaggggccaaacatacctcctgcccatccaggtcaaccctgtcaatgaccc accccac. HMW-MAA-B: (SEQ ID No: 22) Gtccgcgtcactggggccctgcagttttggggagctgcagaaacacggggcaggtggggtggagggtgctgagtggtgggccacacagg cgttccaccagcgggatgtggagcagggccgcgtgaggtacctgagcactgacccacagcaccacgcttacgacaccgtggagaacctggccctgg aggtgcaggtgggccaggagatcctgagcaatctgtccttcccagtgaccatccagagagccactgtgtggatgctgcggctggagccactgcacact cagaacacccagcaggagaccctcaccacagcccacctggaggccaccctggaggaggcaggcccaagccccccaaccttccattatgaggtggtt caggctcccaggaaaggcaaccttcaactacagggcacaaggctgtcagatggccagggcttcacccaggatgacatacaggctggccgggtgacct attggggccacagcacgtgcctcagaggcagtcgaggacaccttccgtttccgtgtcacagctccaccatatttctccccactctataccttccccatcca cattggtggtgacccagatgcgcctgtcctcaccaatgtcctcctcgtggtgcctgagggtggtgagggtgtcctctctgctgaccacctctttgtcaagag tctcaacagtgccagctacctctatgaggtcatggagcggccccgccatgggaggttggcttggcgtgggacacaggacaagaccactatggtgacatcc ttcaccaatgaagacctgttgcgtggccggctggtctaccagcatgatgactccgagaccacagaagatgatatcccatttgttgctacccgccagggcg agagcagtggtgacatggcctgggaggaggtacggggtgtcttccgagtggccatccagcccgtgaatgaccacgcccctgtgcagaccatcagcc ggatcttccatgtggcccggggtgggcggcggctgctgactacagacgacgtggccttcagcgatgctgactcgggctttgctgacgcccagctggtgc ttacccgcaaggacctcctctttggcagtatcgtggccgtagatgagcccacgcggcccatctaccgcttcacccaggaggacctcaggaagaggcga gtactgttcgtgcactcaggggctgaccgtggctggatccagctgcaggtgtccgacgggcaacaccaggccactgcgctgctggaggtgcaggcct cggaaccctacctccgtgtggcc. HMW-MAA-C: (SEQ ID No: 23) Gccactgagccttacaatgctgcccggccctacagcgtggccctgctcagtgtccccgaggccgcccggacggaagcagggaagccag agagcagcacccccacaggcgagccaggccccatggcatccagccctgagcccgctgtggccaagggaggcttcctgagcttccttgaggccaaca tgttcagcgtcatcatccccatgtgcctggtacttctgctcctggcgctcatcctgcccctgctcttctacctccgaaaacgcaacaagacgggcaagcat gacgtccag.

The fragments were amplified using the following primers. The XhoI sites in the forward primers and XmaI sites (A and C) or Spel site (B) in the reverse primers are underlined:

Fragment A:-forward primer (SEQ ID No: 24) TCCTCGAGGTCAATGGCCAGAGGCGGGGG. Reverse: (SEQ ID No: 25) CCCGGGTTACTACTTATCGTCGTCATCCTTGTAATCGTGGGGTGGGTCAT TGAC. Fragment B: forward: (SEQ ID No: 26) GCCTCGAGTTCCGCGTCACTGGGGCCCTG. Reverse: (SEQ ID No: 27) ACTAGTTTACTACTTATCGTCGTCATCCTTGTAATCGGCCACACGGAGGT AGGGTTC. Fragment C: Forward: (SEQ ID No: 28) TGCTCGAGGCCACTGAGCCTTACAATGCTGCC. Reverse: (SEQ ID No: 29) CCCGGGTTACTACTTATCGTCGTCATCCTTGTAATCCTGGACGTCATGCT TGCCCG.

Fragments A-C were then subcloned into pGG-55, using the XhoI site at the end of the hly sequence and the Xmal or Spel site following the gene.

A prfA negative strain of Listeria, XFL-7 (provided by Dr. Hao Shen, University of Pennsylvania), was then transformed with pGG34A, B and C, to select for the retention of the plasmids in vivo.

Example 2 LLO-HMW-MAA Constructs are Expressed in Listeria Materials and Experimental Methods

Bacteria Cultivation and Harvesting

Recombinant Listeria monocytogenes (LM) expressing the HMW-MAA fragments A, B and C fused to LLO were grown overnight in BHI medium supplemented with streptomycin (250 ug/ml) and chloramphenicol (25 ug/ml). For induction of endogeneous LLO, bacteria were cultivated in the presence of 0.2% charcoal. Culture supernatants were cleared by centrifugation at 14000 rpm for 5 minutes, and 1.35 milliliters (ml) supernatant was mixed with 0.15 ml of 100% TCA for protein precipitation. After incubation on ice for 1 hour, the solution was spun for 10 minutes, 14000 rpm. The pellet was resuspended in 45 microliter (mcL) of 1×SDS-PAGE gel loading buffer, 5 mcL of 1 M DTT was added, and the sample was heated at 75° C. for 5 minutes. 5-10 mcL of protein was loaded into each well and run for 50 minutes at 200V using MOPS buffer.

After transfer to PVDF membranes, membranes were incubated with either a rabbit anti-PEST polyclonal antibody (1:3000), which recognizes the PEST sequence in the LLO protein, or with the B3-19 monoclonal antibody, which recognizes the endogenous LLO only, then incubated with HRP-conjugated anti-rabbit antibody. Signals were detected with SuperSignal® West Pico Chemiluminescent Substrate (Pierce, Rockford, Ill.).

Results

To determine whether the LLO-HMW-MAA constructs could be expressed in Listeria, supernatant was harvested from LM strains transformed with the LLO-HMW-MAA A, B and C plasmids, and assayed for presence of the fusion proteins. All three strains produced fusion proteins of the expected sizes when probed with anti-PEST antibody (48 Kda for LLO, 75 Kda for HMW-MAA-A, 98 Kda for HMW-MAA-B, and 62 Kda for HMW-MAA-C; FIG. 2A). Anti-LLO antibody revealed 58 Kda band for LLO in all three strains and controls (FIG. 2B).

Thus, LLO-HMW-MAA constructs are expressed in Listeria.

Example 3 Listeria Strains Expressing LLO-HMW-MAA Constructs Infect and Grow Inside Cells Materials and Experimental Methods

Cell Infection Assay

Murine macrophage-like J774 cells were infected at a MOI (multiplicity of infection) of 1. After a 1-hour incubation, gentamicin was added to kill extracellular Listeria, intracellular Listeria was recovered every 2 hours by lysing the J774 cells with water and plating serial dilutions of the lysate on BHI plates supplemented with streptomycin (250 micrograms (mcg)/ml) and chloramphenicol (25 mcg/ml). Recovered colonies were counted and used to determine the number of Listeria inside J774 cells.

Results

To determine the growth characteristics and virulence of Listeria strains expressing LLO-HMW-MAA constructs, the growth rate of Listeria strains from the previous Example in BHI media was measured. Each of the strains grew with kinetics very similar to wild-type (1043S) Listeria (FIG. 3A). Next, J774 cells were incubated with the Listeria strains, and intracellular growth was measured. Intracellular growth was very similar to wild-type for each strain (FIG. 3B).

Thus, Listeria strains expressing LLO-HMW-MAA constructs maintain their ability to grow in media, to infect cells, and to grow intracellularly.

Example 4 Vaccination with HMW-MAA-Expressing Lm Impedes B16F0-OVA Tumor Growth Materials and Experimental Methods

Measurement of Tumor Growth

Tumors were measured every second day with calipers spanning the shortest and longest surface diameters. The mean of these two measurements was plotted as the mean tumor diameter in millimeters against various time points. Mice were sacrificed when the tumor diameter reached 20 mm. Tumor measurements for each time point are shown only for surviving mice.

Results

32 C57BL/6 mice (n=8 per group) were inoculated with 5×10⁵ B16F0-Ova. On days 3, 10 and 17 the mice were immunized with one of3 constructs, Lm-OVA (10⁶ cfu), Lm-LLO-OVA (10⁸ cfu; positive control), and Lm-LLO-HMW-MAA-C (10⁸ cfu). Despite the lack of expression of HMW-MAA by the tumor cells, Lm-LLO-HMW-MAA-C vaccination impeded tumor growth, significantly, but to a lesser extent than Lm-LLO-OVA (FIGS. 4A-B). In an additional experiment, similar results were observed with all 3 Lm-LLO-HMW-MAA strains (2.5×10⁷ cfu each of A and C; 1×10⁸ cfu of B; FIG. 4C).

A similar experiment was performed with RENCA cells. 40 BALB/c mice (n=8 per group) were inoculated with 2×10⁵ RENCA tumor cells. On days 3, 10, and 17, the mice were immunized with one of four constructs, Lm-HMW-MAA-A, B, or C ((2.5×10⁷ cfu each of A and C; 1×10⁸ cfu of B), or GGE7 (Lm-LLO-E7; 1.0×10⁸ cfu), or were left unvaccinated (naïve). All three Lm-LLO-HMW-MAA strains impeded tumor growth, with Lm-HMW-MAA-C exerting the strongest effect (FIGS. 4D-4E).

Thus, vaccination with HMW-MAA-expressing Lm impedes the growth of tumors, even in the absence of expression of HMW-MAA by the tumor cells.

Example 5 Vaccination with HMW-MAA-Expressing Lm Impedes B16F10-HMW-MAA Tumor Growth Via Cd4+ and Cd8+ Cells Materials and Experimental Methods

Engineering of B16 and B16F10 Murine Tumor Cell Lines to Express HMW-MAA

B16F10 cells were transfected with pcDNA3.1-HMW-MAA plasmid, containing the full-length HMW-MAA cDNA expressed under the control of the CMV promoter. Stably transfected cells were selected by resistance to G418 antibiotic, and clones were subsequently grown from single cells by limiting dilution. Selected clones were tested by flow cytometry for HMW-MAA expression using the HMW-MAA specific monoclonal antibody VT80.12. Based on the flow cytometry results, B16F10-HMW-MAA clone 7 was selected for future experiments (FIG. 5).

CD4+ and CD8+ Depletion

32 C57BL/6 mice were inoculated with 2×10⁵ B16F10-HMW-MAA/CMV7. On days 3, 10 and 17 the mice were immunized with Lm-HMW-MAA-C (2.5×10⁷ cfu), except the control naïve group. For CD4 and CD8 depletions, 500 μg of GK1.5 and 2.43 were given i.p. on days 1, 2, 6 and 9, as well as the control antibody G1. For CD25 depletion, 500 μg of CP61 was given i.p. on days 0 and 2.

Immunization of HLA A2/K^(b) Transgenic Mice with Lm-HMW-MAA-B or Lm-HMW-MAA-C

HLA A2/K^(b) transgenic mice express a chimeric class I molecule composed of the α1 and α2 domains of the human A*0201 allele and the α3 domains of the mouse H-2K^(b) class I molecules. HLA-A2/K^(b) transgenic mice were immunized once with either 1.0×10⁸ cfu of Lm-HMW-MAA-B or 2.5×10⁷ cfu of Lm-HMW-MAA-C. 9 days later, splenocytes were stimulated in vitro with peptide B₁ (ILSNLSFPV; SEQ ID NO: 43; corresponds to HMW-MAA₇₆₉₋₇₇₇), peptide B₂ (LLFGSIVAV; SEQ ID NO: 44; corresponds to HMW-MAA₁₀₆₃₋₁₀₇₁), or peptide C (LILPLLFYL; SEQ ID NO: 45; corresponds to HMW-MAA₂₂₃₈₋₂₂₄₆) for 5 hours in the presence of monensin. Cells were gated on CD8+-CD62L^(low) and IFN-γ intracellular staining was measured.

In a separate experiment, mice were immunized twice (day 0 and day 7) with either Lm-HMW-MAA-B or Lm-HMW-MAA-C and splenocytes harvested on day 14 for in vitro stimulation with fragment B1 or C of Lm-HMW-MAA. IFN-γ levels were measured using IFN-γ Elispot.

Results

C57BL/6 mice (n=8 per group) were inoculated with 2×10⁵ B16F10-HMW-MAA/CMV7. On days 3, 10 and 17, mice were immunized with one of three constructs, Lm-HMW-MAA-A (2.5×10⁷ cfu), Lm-HMW-MAA-B (1×10⁸ cfu), Lm-HMW-MAA-C (2.5×10⁷ cfu). The control group was vaccinated with Lm-GGE7 (1×10⁸ cfu). All three Lm-HMW-MAA constructs exerted significant anti-tumor effects (FIG. 6).

To determine the role of CD4+ and CD8+ cells in the anti-tumor effect of Lm-HMW-MAA, CD4+ or CD8+, cells were depleted in C57BL/6 mice who had been innoculated with B16F10-HMW-MAA/CMV7B and immunized on days 3, 10, and 17 with Lm-HMW-MAA-C (2.5×10⁷ cfu). CD4+ or CD8+ depletion abrogated the efficacy of LM-HMW-MAA-C vaccine (FIG. 7).

CD8+ T cells (2×10⁶ cells per mouse) were purified from the spleens of mice from each treatment group, mixed with B16F10-HMW-MAA tumor cells (2×10⁵ per mouse), and then subcutaneously injected in mice (8 per group). Mice were observed for 28 days and examined every 2 days for tumor growth. CD8+ T cells from Lm-HMW-MAA-C-vaccinated mice inhibited the growth of B 16F10 HMW-MAA tumors in vivo (FIG. 8).

Mice that had been inoculated with 2×10⁵ B 16F10-HMW-MAA/CMV7 and vaccinated with Lm-HMW-MAA-C as described above and remained tumor-free after 7 weeks were re-challenged with 2×10⁵ B16F10-HMW-MAA cells 7 weeks after the first tumor injection. Vaccinated mice were protected against a second challenge with B16F10-HMW-MAA/CMV7 tumor cells (FIG. 9).

Immunization of HLA-A2/K^(b) transgenic mice with Lm-HMW-MAA-B and Lm-HMW-MAA-C induces detectable immune responses against two characterized HMW-MAA HLA-A2 epitopes in fragments B and C both after one (FIG. 10A) or two immunizations (FIG. 10B).

HLA-A2/K^(b) and wild-type C57B1/6 mice were immunized once with Lm-HMW-MAA-B or Lm-HMW-MAA-C, and IFN-γ secretion by T cells stimulated with an HLA-A2 restricted peptide from fragment C was measured with IFN-γ Elispot. IFN-γ secretion was increased in Lm-HMW-MAA-C-immunized HLA-A2/K^(b) transgenic mice stimulated with Peptide C compared to unstimulated transgenic mice, compared to Peptide C-stimulated non-transgenic mice and compared to non-immunized transgenic and control mice (FIGS. 11A and 11B).

Thus, Lm-HMW-MAA constructs induce antigen-specific immune responses that impede tumor growth. In addition, the Lm-HMW-MAA constructs exhibit anti-tumor activity even against tumors not expressing HMW-MAA.

Example 6 Fusion oF E7 to LLO or ActA Enhances E7-Specific Immunity and Generates Tumor-Infiltrating E7-Specific CD8⁺ Cells Materials and Experimental Methods

Construction of Lm-actA-E7

Lm-actA-E7 was generated by introducing a plasmid vector pDD-1 constructed by modifying pDP-2028 into LM. pDD-1 comprises an expression cassette expressing a copy of the 310 bp hly promoter and the hly signal sequence (ss), which drives the expression and secretion of actA-E7; 1170 bp of the actA gene that comprises 4 PEST sequences (SEQ ID NO: 5) (the truncated ActA polypeptide consists of the first 390 AA of the molecule, SEQ ID NO: 4); the 300 bp HPV E7 gene; the 1019 bp prfA gene (controls expression of the virulence genes); and the CAT gene (chloramphenicol resistance gene) for selection of transformed bacteria clones.

pDD-1 was created from pDP2028 (encoding ALLO-NP), which was in turn created from pDP1659 as follows:

Construction of pDP1659: The DNA fragment encoding the first 420 AA of LLO and its promoter and upstream regulatory sequences was PCR amplified with LM genomic DNA used as a template and ligated into pUC19. PCR primers used were 5′-GGCCCGGGCCCCCTCCTTTGAT-3′ (SEQ ID No: 30) and 5′-GGTCTAGATCATAATTTACTTCATCC-3′ (SEQ ID No: 31). The DNA fragment encoding NP was similarly PCR amplified from linearized plasmid pAPR501 (obtained from Dr. Peter Palese, Mt. Sinai Medical School, New York) and subsequently ligated as an in-frame translational fusion into pUC19 downstream of the hemolysin gene fragment. PCR primers used were 5′-GGTCTAGAGAATTCCAGCAAAAGCAG-3′ (SEQ ID No: 32) and 5′-GGGTCGACAAGGGTATTTTTCTTTAAT-3′ (SEQ ID No: 33). The fusion was then subcloned into the EcoRV and SalI sites of pAM401. Plasmid pDP2028 was constructed by subcloning the prfA gene into the SalI site of pDP1659.

pDD-1 was created from pDP-2028 (Lm-LLO-NP) as follows:

The hly promoter (pHly) and gene fragment (441 AA) were PCR amplified from pGG55 using primer 5′-GGGGTCTAGACCTCCTTTGATTAGTATATTC-3′ (XbaI site is underlined; SEQ ID NO: 34) and primer 5′-ATCTTCGCTATCTGTCGCCGCGGCGCGTGCTTCAGTTTGTTGCGC-′3 (Not I site is underlined. The first 18 nucleotides are the ActA gene overlap; SEQ ID NO: 35). The actA gene was PCR amplified from the LM 10403s wildtype genome using primer 5′-GCGCAACAAACTGAAGCAGCGGCCGCGGCGACAGATAGCGAAGAT-3′ (NotI site is underlined; SEQ ID NO: 36) and primer 5′-TGTAGGTGTATCTCCATGCTCGAGAGCTAGGCGATCAATTTC-3′ (XhoI site is underlined; SEQ ID NO: 37). The E7 gene was PCR amplified from pGG55 using primer 5′-GGAATTGATCGCCTAGCTCTCGAGCATGGAGATACACCTACA-3′ (XhoI site is underlined; SEQ ID NO: 38) and primer 5′-AAACGGATTTATTTAGATCCCGGGTTATGGTTTCTGAGAACA-3′ (XmaI site is underlined; SEQ ID NO: 39). The prfA gene was PCR amplified from the LM 10403s wild-type genome using primer 5′-TGTTCTCAGAAACCATAACCCGGGATCTAAATAAATCCGTTT-3′ (XmaI site is underlined; SEQ ID NO: 40) and primer 5′-GGGGGTCGACCAGCTCTTCTTGGTGAAG-3′ (SalI site is underlined; SEQ ID NO: 41). The hly promoter-actA gene fusion (pHly-actA) was PCR generated and amplified from purified pHly and actA DNA using the upstream pHly primer (SEQ ID NO: 34) and downstream actA primer (SEQ ID NO: 37).

The E7 gene fused to the prfA gene (E7-prfA) was PCR generated and amplified from purified E7 and prfA DNA using the upstream E7 primer (SEQ ID NO: 38) and downstream prfA gene primer (SEQ ID NO: 41).

The pHly-actA fusion product fused to the E7-prfA fusion product was PCR generated and amplified from purified fused pHly-actA and E7-prfA DNA products using the upstream pHly primer (SEQ ID NO: 34) and downstream prfA gene primer (SEQ ID NO: 41) and ligated into pCRII (Invitrogen, La Jolla, Calif.). Competent E. coli (TOP10′F, Invitrogen, La Jolla, Calif.) were transformed with pCRII-ActAE7. After lysis and isolation, the plasmid was screened by restriction analysis using BamHI (expected fragment sizes 770 and 6400 bp) and BstXI (expected fragment sizes 2800 and 3900) and screened by PCR using the above-described upstream pHly primer and downstream prfA gene primer.

The pHly-ActA-E7-PrfA DNA insert was excised from pCRII by XbaI/SalI digestion with and ligated into Xba I/Sal I digested pDP-2028. After transforming TOP10′F competent E. coli (Invitrogen, La Jolla, Calif.) with expression system pHly-ActA-E7, chloramphenicol resistant clones were screened by PCR analysis using the above-described upstream pHly primer and downstream prfA gene primer. A clone containing pHly-ActA-E7 was amplified, and midiprep DNA was isolated (Promega, Madison, Wis). XFL-7 was transformed with pHly-ActA-E7, and clones were selected for the retention of the plasmid in vivo. Clones were grown in brain heart infusion medium (Difco, Detroit, Mich.) with 20 mcg (microgram)/ml (milliliter) chloramphenicol at 37° C. Bacteria were frozen in aliquots at −80° C.

In vivo Experiments

500 mcL of MATRIGEL®, containing 100 mcL of phosphate buffered saline (PBS) with 2×10⁵ TC-1 tumor cells, plus 400 mcL of Matrigel® (BD Biosciences, Franklin Lakes, N.J.) were implanted subcutaneously on the left flank of 12 C57BL/6 mice (n=3). Mice were immunized intraperitoneally on day 7, 14 and 21, and spleens and tumors were harvested on day 28. Tumor Matrigels were removed from the mice and incubated at 4° C. overnight in tubes containing 2 ml RP 10 medium on ice. Tumors were minced.with forceps, cut into 2 mm blocks, and incubated at 37° C. for 1 hour with 3 ml of enzyme mixture (0.2 mg/ml collagenase-P, 1 mg/ml DNAse-1 in PBS). The tissue suspension was filtered through nylon mesh and washed with 5% fetal bovine serum+0.05% of NaN₃ in PBS for tetramer and IFN-gamma staining.

Splenocytes and tumor cells were incubated with 1 micromole (mcm) E7 peptide for 5 hours in the presence of brefeldin A at 10⁷ cells/ml. Cells were washed twice and incubated in 50 mcL of anti-mouse Fc receptor supernatant (2.4 G2) for 1 hour or overnight at 4° C. Cells were stained for surface molecules CD8 and CD62L, permeabilized, fixed using the permeabilization kit Golgi-stop® or Golgi-Plug® (Pharmingen, San Diego, Calif.), and stained for IFN-gamma. 500,000 events were acquired using two-laser flow cytometer FACSCalibur and analyzed using Cellquest Software (Becton Dickinson, Franklin Lakes, N.J.). Percentages of IFN-gamma secreting cells within the activated (CD62L^(low)) CD8⁺ T cells were calculated.

For tetramer staining, H-2D^(b) tetramer was loaded with phycoerythrin (PE)-conjugated E7 peptide (RAHYNIVTF, SEQ ID NO: 42), stained at rt for 1 hour, and stained with anti-allophycocyanin (APC) conjugated MEL-14 (CD62L) and FITC-conjugated CD8β at 4° C. for 30 min. Cells were analyzed comparing tetramer⁺ CD8⁺ CD62L^(low) cells in the spleen and in the tumor.

Results

To analyze the ability of LLO and ActA fusions to enhance antigen specific immunity, mice were implanted with TC-1 tumor cells and immunized with either Lm-LLO-E7 (1×10⁷ CFU), Lm-E7 (1×10⁶ CFU), or Lm-ActA-E7 (2×10⁸ CFU), or were untreated (naïve). Tumors of mice from the Lm-LLO-E7 and Lm-ActA-E7 groups contained a higher percentage of IFN-gamma-secreting CD8⁺ T cells (FIG. 12) and tetramer-specific CD8⁺ cells (FIG. 13) than in mice administered Lm-E7 or naive mice. Thus, Lm-LLO-E7 and Lm-ActA-E7 are both efficacious at induction of tumor-infiltrating CD8⁺ T cells and tumor regression. Accordingly, LLO and ActA fusions are effective in methods and compositions of the present invention.

Example 7 Fusion to a PEST-Like Sequence Enhances E7-Specific Immunity Materials and Experimental Methods

Constructs

Lm-PEST-E7, a Listeria strain identical to Lm-LLO-E7, except that it contains only the promoter and the first 50 AA of the LLO, was constructed as follows:

The hly promoter and PEST regions were fused to the full-length E7 gene by splicing by overlap extension (SOE) PCR. The E7 gene and the hly-PEST gene fragment were amplified from the plasmid pGG-55, which contains the first 441 amino acids of LLO, and spliced together by conventional PCR techniques. pVS16.5, the hly-PEST-E7 fragment and the LM transcription factor prfA were subcloned into the plasmid pAM401. The resultant plasmid was used to transform XFL-7.

Lm-E7_(epi) is a recombinant strain that secretes E7 without the PEST region or an LLO fragment. The plasmid used to transform this strain contains a gene fragment of the hly promoter and signal sequence fused to the E7 gene. This construct differs from the original Lm-E7, which expressed a single copy of the E7 gene integrated into the chromosome. Lm-E7_(epi) is completely isogenic to Lm-LLO-E7 and Lm-PEST-E7, except for the form of the E7 antigen expressed.

Recombinant strains were grown in brain heart infusion (BHI) medium with chloramphenicol (20 mcg/mL). Bacteria were frozen in aliquots at −80° C.

Results

To test the effect on antigenicity of fusion to a PEST-like sequence, the LLO PEST-like sequence was fused to E7. Tumor regression studies were performed, as described for Example 1, in parallel with Listeria strain expressing LLO-E7 and E7 alone. Lm-LLO-E7 and Lm-PEST-E7 caused the regression ⅝ and ⅜ established tumors, respectively (FIG. 14). By contrast, Lm-E7epi only caused tumor regression in ⅛ mice. A statistically significant difference in tumor sizes was observed between tumors treated with PEST-containing constructs (Lm-LLO-E7 or Lm-PEST-E7) and those treated with Lm-E7epi (Student's t test).

To compare the levels of E7-specific lymphocytes generated by the vaccines in the spleen, spleens were harvested on day 21 and stained with antibodies to CD62L, CD8, and the E7/Db tetramer. Lm-E7_(epi) induced low levels of E7 tetramer-positive activated CD8⁺ T cells in the spleen, while Lm-PEST-E7 and Lm-LLO-E7 induced 5 and 15 times more cells, respectively (FIG. 15A), a result that was reproducible over 3 separate experiments. Thus, fusion to PEST-like sequences increased induction of tetramer-positive splenocytes. The mean and SE of data obtained from the 3 experiments (FIG. 15B) demonstrate the significant increase in tetramer-positive CD8⁺ cells by Lm-LLO-E7 and Lm-PEST-E7 overLm-E7epi (P<0.05 by Student's t test). Similarly, the number of tumor-infiltrating antigen-specific CD8⁺ T cells was higher in mice vaccinated with Lm-LLO-E7 and Lm-PEST-E7, reproducibly over 3 experiments (FIG. 16A-B). Average values of tetramer-positive CD8⁺ TILs were significantly higher for Lm-LLO-E7 than Lm-E7epi (P<0.05; Student's t test.

Thus, PEST-like sequences confer increased immunogenicity to antigens.

Example 8 Enhancement of Immunogenicity by Fusion of an Antigen to LLO Does Not Require a Listeria Vector Materials and Experimental Methods

Construction of Vac-SigE7Lamp

The WR strain of vaccinia was used as the recipient, and the fusion gene was excised from the Listerial plasmid and inserted into pSC11 under the control of the p75 promoter. This vector was chosen because it is the transfer vector used for the vaccinia constructs Vac-SigE7Lamp and Vac-E7 and therefore allowed direct comparison with Vac-LLO-E7. In this way all 3 vaccinia recombinants were expressed under control of the same early/late compound promoter p7.5. In addition, SC11 allows the selection of recombinant viral plaques to TK selection and beta-galactosidase screening. FIG. 17 depicts the various vaccinia constructs used in these experiments. Vac-SigE7Lamp is a recombinant vaccinia virus that expressed the E7 protein fused between lysosomal associated membrane protein (LAMP-1) signal sequence and sequence from the cytoplasmic tail of LAMP-1.

The following modifications were made to allow expression of the gene product by vaccinia: (a) the T5XT sequence that prevents early transcription by vaccinia was removed from the 5′ portion of the LLO-E7 sequence by PCR; and (b) an additional XmaI restriction site was introduced by PCR to allow the final insertion of LLO-E7 into SC11. Successful introduction of these changes (without loss of the original sequence that encodes for LLO-E7) was verified by sequencing. The resulting pSC1 1-E7 construct was used to transfect the TK-ve cell line CV1 that had been infected with the wild-type vaccinia strain, WR. Cell lysates obtained from this co-infection/transfection step contain vaccinia recombinants that were plaque-purified 3 times. Expression of the LLO-E7 fusion product by plaque-purified vaccinia was verified by Western blot using an antibody directed against the LLO protein sequence. Ability of Vac-LLO-E7 to produce CD8⁺ T cells specific to LLO and E7 was determined using the LLO (91-99) and E7 (49-57) epitopes of Balb/c and C57/BL6 mice, respectively. Results were confirmed in a chromium release assay.

Results

To determine whether enhancement of immunogenicity by fusion of an antigen to LLO requires a Listeria vector, a vaccinia vector expressing E7 as a fusion protein with a non-hemolytic truncated form of LLO was constructed. Tumor rejection studies were performed with TC-1 as described in above Examples, but initiating treatment when the tumors were 3 mm in diameter (FIG. 18). By day 76, 50% of the Vac-LLO-E7 treated mice were tumor free, while only 25% of the Vac-SigE7Lamp mice were tumor free. In other experiments, LLO-antigen fusions were shown to be more immunogenic than E7 peptide mixed with SBAS2 or unmethylated CpG oligonucleotides in a side-by-side comparison.

These results show that (a) LLO-antigen fusions are immunogenic not only in the context of Listeria, but also in other contexts; and (b) the immunogenicity of LLO-antigen fusions compares favorably with other vaccine approaches known to be efficacious. 

1. A recombinant Listeria strain comprising a recombinant nucleotide sequence encoding a recombinant polypeptide, said recombinant polypeptide comprising a fragment of a High Molecular Weight Melanoma-Associated Antigen (HMW-MAA) protein fused to a listeriolysin (LLO) oligopeptide, an ActA oligopeptide, or a PEST oligopeptide, wherein said nucleotide sequence encoding said fragment is selected from the group consisting of SEQ ID NOs: 21, 22 or
 23. 2. The recombinant Listeria strain of claim 1, wherein said HMW-MAA protein is a human HMW-MAA protein.
 3. An immunogenic composition comprising the recombinant Listeria strain of claim 1, or claims 5-7.
 4. The immunogenic composition of claim 3, further comprising an adjuvant, cytokine, chemokine, or combination thereof.
 5. A recombinant Listeria strain comprising a recombinant nucleotide sequence encoding a recombinant polypeptide, said recombinant polypeptide comprising a fragment of a High Molecular Weight Melanoma-Associated Antigen (HMW-MAA) protein fused to a listeriolysin (LLO) oligopeptide, an ActA oligopeptide, or a PEST oligopeptide, wherein said nucleotide sequence encodes SEQ ID NO:
 21. 6. A recombinant Listeria strain comprising a recombinant nucleotide sequence encoding a recombinant polypeptide, said recombinant polypeptide comprising a fragment of a High Molecular Weight Melanoma-Associated Antigen (HMW-MAA) protein fused to a listeriolysin (LLO) oligopeptide, an ActA oligopeptide, or a PEST oligopeptide, wherein said nucleotide sequence encodes SEQ ID NO:
 22. 7. A recombinant Listeria strain comprising a recombinant nucleotide sequence encoding a recombinant polypeptide, said recombinant polypeptide comprising a fragment of a High Molecular Weight Melanoma-Associated Antigen (HMW-MAA) protein fused to a listeriolysin (LLO) oligopeptide, an ActA oligopeptide, or a PEST oligopeptide, wherein said nucleotide sequence encodes SEQ ID NO:
 23. 8. The recombinant Listeria strain of claim 1, wherein said recombinant Listeria strain is a recombinant Listeria monocytogenes strain.
 9. The recombinant Listeria strain of claim 1, wherein said recombinant Listeria strain has been passaged through an animal host. 